Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo methods
Aim. Evidence-backed execution summary for Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo methods from Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo.
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mouse
Subject model for the experiment.
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ORs and OR83b Adopt a Novel Membrane Topology
reagent used in the protocol.
- Use
- (C) Salivary glands expressing GFP:OR83b ( AB1-Gal4/+;UAS-GFP:Or83b/+ ) were stained with antibodies against the epitopes, illustrated in red in the snake plots on the left, under permeabilized or unpermeabilized conditions. For clarity, only the red channel is shown. None of the antibodies stain control salivary gl...
ORs and OR83b Form Heteromeric Complexes in the Sensory Cilia of OSNs
reagent used in the protocol.
- Use
- We next asked whether this functional odorant receptor is composed of a complex of OR and OR83b proteins that is present in vivo at the site of odor detection ( ). Previous in vitro efforts suggested that ORs can form both homomers and heteromers with OR83b, but no evidence was offered that these complexes are funct...
ORs and OR83b Adopt a Novel Membrane Topology
reagent used in the protocol.
- Use
- (E) ImmunoEM on a horizontal section of an antennal sensillum using OR83b α-EC2 and a secondary antibody conjugated to 5 nm colloidal gold reveals distribution of the EC2 epitope on the extracellular face of the dendritic membranes (scale bar = 200 nm).
ORs and OR83b Adopt a Novel Membrane Topology
reagent used in the protocol.
- Use
- To obtain experimental evidence that TM1 of Drosophila ORs inserts into the membrane with the N-terminus intracellular, we first used the β-galactosidase β-gal fusion technique. This method takes advantage of the observation that β-gal is enzymatically active when present in the cytosolic compartment...
ORs and OR83b Adopt a Novel Membrane Topology
reagent used in the protocol.
- Use
- We generated constructs encoding either the N-terminal domain alone or the N-terminal domain and TM1 of OR83b fused at their C-termini to β-gal. As a control, we generated constructs in which a synthetic TM domain was placed between the fragments of OR83b and β-gal, which are predicted to give opposite res...
ORs and OR83b Adopt a Novel Membrane Topology
reagent used in the protocol.
- Use
- To examine OR topology beyond the location of the N-terminus, we performed OR83b antibody epitope-staining experiments, which probe topology by comparing antibody access in permeabilized and non-permeabilized conditions. OSN dendrites proved to be inaccessible to labeling without compromising cell permeability. We f...
ORs and OR83b Adopt a Novel Membrane Topology
reagent used in the protocol.
- Use
- (A) Left panel: whole-mount view of a third instar larval salivary gland expressing GFP:OR83b (green) counterstained with DAPI (blue) to visualize the cell nuclei. Genotype in this and subsequent panels: AB1-Gal4/+;UAS-GFP:Or83b/+. The white box marks the approximate field of view of this tissue shown in all subseq...
ORs and OR83b Associate via Conserved Cytoplasmic C-Terminal Domains
reagent used in the protocol.
- Use
- (B) Interactions between OR83b and OR cytoplasmic domains detected by the yeast two-hybrid assay by observation of growth (+) or no growth (-) of yeast co-transformed with the indicated bait/prey combinations on media selecting for expression of HIS3 and ADE2 reporters.
OR83b Is Continuously Required for OR Localization
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction o...
- Use
- To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction o...
OR83b Is Continuously Required for OR Localization
(A) Schematic of the TARGET system.
- Use
- (A) Schematic of the TARGET system.
OR83b Is Continuously Required for OR Localization
(B) Immunostaining for OR83b (green) and OR22a/b (red) in Or83b null-mutant flies rescued using a UAS-Or83b transgene under the control of the TARGET system ( Or22a-Gal4,tubP-Gal80ts/tubP-Gal80ts;UAS-Or83b,Or83b 1 /tubP-Gal80ts,Or83b 2 ). Three copies of the tubP-Gal80ts transgene were used to ensure efficient suppr...
- Use
- (B) Immunostaining for OR83b (green) and OR22a/b (red) in Or83b null-mutant flies rescued using a UAS-Or83b transgene under the control of the TARGET system ( Or22a-Gal4,tubP-Gal80ts/tubP-Gal80ts;UAS-Or83b,Or83b 1 /tubP-Gal80ts,Or83b 2 ). Three copies of the tubP-Gal80ts transgene were used to ensure efficient suppr...
Histology
Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyd...
- Use
- Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyd...
Optical imaging
In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames...
- Use
- In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames...
Topology-mapping with β-gal fusion proteins
lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41,...
- Use
- lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41,...
Electron microscopy
Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing...
- Use
- Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing...
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ORs and OR83b Adopt a Novel Membrane Topology
(C) Salivary glands expressing GFP:OR83b ( AB1-Gal4/+;UAS-GFP:Or83b/+ ) were stained with antibodies against the epitopes, illustrated in red in the snake plots on the left, under permeabilized or unpermeabilized conditions. For clarity, only the red channel is shown. None of the antibodies stain control salivary glands under permeabilized conditions (unpublished data).
OR83b Is Continuously Required for OR Localization
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction of a UAS-Or83b transgene by an Or22a-Gal4 driver line with temporal regulation through use of a ubiquitously expressed temperature-sensitive Gal80 (Gal80ts) transgene. At the permissive temperature (18 °C), the GAL80 protein is active and represses induction of OR83b expression by inhibiting GAL4 ( A, top row). At the restrictive temperature (29 °C), GAL80 is inactivated, permitting expression of OR83b in OR22a/b neurons ( A, bottom row).
Histology
Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyde in PBS for 20 min. These were then stained as for antennal sections, either in the presence of 0.25% Triton X-100 (Fisher Scientific, Springfield, New Jersey, United States) (permeabilized) or without detergent (non-permeabilized). After mounting in Vectashield (Vector Labs, Burlingame, California, United States), salivary glands were dissected away from other tissues. Images were collected with a Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany).
Optical imaging
In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames was taken at 4 Hz. A constant air stream (2,000 ml/min) produced by an aquarium pump was guided through a 1-ml syringe with the tip placed 1 cm from the antennae. Pure odorants were diluted in paraffin oil (see figure legends for concentrations), and 10 µl of this solution was placed on a filter paper in a second syringe that was laterally inserted into this air flow. Odorant stimuli (1 s, i.e., frames 12-16) were puffed into the constant airstream using a custom-made electronic valve under computer control with a 1-min interstimulus interval. Imaging data were...
Topology-mapping with β-gal fusion proteins
lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41, 1-68), RH1 (1-49, 1-82). Plasmids were transfected into S2-R+ cultured Drosophila cells [ ] with Fugene (Qiagen, Valencia, California, United States). Expression of fusion proteins was induced for 24 h with 5 mM Cu 2 S0 4 and verified by Western blotting of cell extracts using antibodies against β-gal (Cappel from MP Biomedicals, Irvine, California, United States). To assess β-gal activity, cells were rinsed in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min, rinsed twice in PBS, and incubated in staining solution [5 mM K 3 Fe(...
Electron microscopy
Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing with PBS, the sections were incubated with goat-α-rabbit conjugated to 5 nm gold particles (Amersham Biosciences, Little Chalfont, United Kingdom). Incubations were stopped by buffer washes followed by fixation with 2.5% glutaraldehyde. Sections were further processed as described above except embedding was in EPON. Silver sections were collected on copper grids and viewed unstained in a JEOL 100 CX electron microscope operated at 80 kV. Distributions of gold particles were scored blindly by an impartial observer.
Measurement outputs
What raw and processed outputs should exist?
(C) Immunostaining for OR83b (green) and OR22a/b (red) in flies [genotype as in (B)] that were cultured and aged as adults for 3 d at 29 °C, transferred to 18 °C to sw...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
(B) Immunostaining for OR83b (green) and OR22a/b (red) in Or83b null-mutant flies rescued using a UAS-Or83b transgene under the control of the TARGET system ( Or22a-Gal4,tubP-Ga...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
In the first experiment, we cultured flies at 18 °C, collected adults, and aged these for 10 d at 18 °C. We then split these animals into two groups and incubated them...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
In flies expressing only G-CaMP, robust responses are observed, as expected, to CO 2, but not to cyclohexanol, a known OR43a ligand [, ] ( A, left column).
from paperScoring or quantification
Quantify the primary readouts for this experiment: (C) Immunostaining for OR83b (green) and OR22a/b (red) in flies [genotype as in (B)] that were cultured and aged as adults for 3 d at 29 °C, transferred to 18 °C to sw...; To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system...; (B) Immunostaining for OR83b (green) and OR22a/b (red) in Or83b null-mutant flies rescued using a UAS-Or83b transgene under the control of the TARGET system ( Or22a-Gal4,tubP-Ga...; In the first experiment, we cultured flies at 18 °C, collected adults, and aged these for 10 d at 18 °C. We then split these animals into two groups and incubated them....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
In flies expressing only G-CaMP, robust responses are observed, as expected, to CO 2, but not to cyclohexanol, a known OR43a ligand [, ] ( A, left column). When GFP:OR43a and...; We investigated whether these heteromeric complexes are functional by expressing these fusion proteins along with G-CaMP in Or83b mutant neurons and assessing odor-evoked calciu...; To define the regions that mediate the specific association of ORs with OR83b, we initiated a structure/function analysis of these receptors. This was initially constrained by t...
from paperReporting output
Report representative outputs alongside summary comparisons for (C) Immunostaining for OR83b (green) and OR22a/b (red) in flies [genotype as in (B)] that were cultured and aged as adults for 3 d at 29 °C, transferred to 18 °C to sw..., To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system..., (B) Immunostaining for OR83b (green) and OR22a/b (red) in Or83b null-mutant flies rescued using a UAS-Or83b transgene under the control of the TARGET system ( Or22a-Gal4,tubP-Ga..., In the first experiment, we cultured flies at 18 °C, collected adults, and aged these for 10 d at 18 °C. We then split these animals into two groups and incubated them....
inferred from protocolStructured statistical methods
In flies expressing only G-CaMP, robust responses are observed, as expected, to CO 2, but not to cyclohexanol, a known OR43a ligand [, ] ( A, left column). When GFP:OR43a and...; We investigated whether these heteromeric complexes are functional by expressing these fusion proteins along with G-CaMP in Or83b mutant neurons and assessing odor-evoked calciu...; To define the regions that mediate the specific association of ORs with OR83b, we initiated a structure/function analysis of these receptors. This was initially constrained by t...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
(C) Salivary glands expressing GFP:OR83b ( AB1-Gal4/+;UAS-GFP:Or83b/+ ) were stained with antibodies against the epitopes, illustrated in red in the snake plots on the left, under permeabilized or unpermeabilized conditions. For clarity, only the red channel is shown. None of the antibodies stain control salivary glands under permeabilized conditions (unpublished data).
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction of a UAS-Or83b transgene by an Or22a-Gal4 driver line with temporal regulation through use of a ubiquitously expressed temperature-sensitive Gal80 (Gal80ts) transgene. At the permissive temperature (18 °C), the GAL80 protein is active and represses induction of OR83b expression by inhibiting GAL4 ( A, top row). At the restrictive temperature (29 °C), GAL80 is inactivated, permitting expression of OR83b in OR22a/b neurons ( A, bottom row).
Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyde in PBS for 20 min. These were then stained as for antennal sections, either in the presence of 0.25% Triton X-100 (Fisher Scientific, Springfield, New Jersey, United States) (permeabilized) or without detergent (non-permeabilized). After mounting in Vectashield (Vector Labs, Burlingame, California, United States), salivary glands were dissected away from other tissues. Images were collected with a Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany).
In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames was taken at 4 Hz. A constant air stream (2,000 ml/min) produced by an aquarium pump was guided through a 1-ml syringe with the tip placed 1 cm from the antennae. Pure odorants were diluted in paraffin oil (see figure legends for concentrations), and 10 µl of this solution was placed on a filter paper in a second syringe that was laterally inserted into this air flow. Odorant stimuli (1 s, i.e., frames 12-16) were puffed into the constant airstream using a custom-made electronic valve under computer control with a 1-min interstimulus interval. Imaging data were analyzed with custom-written IDL software (Research Systems, Boulder, Colorado, United States) including noise filtering, movement, and bleaching correction. Relative fluorescence changes (ΔF/F) were calculated by subtracting the averaged fluorescence intensities from frames 4-6 from the...
lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41, 1-68), RH1 (1-49, 1-82). Plasmids were transfected into S2-R+ cultured Drosophila cells [ ] with Fugene (Qiagen, Valencia, California, United States). Expression of fusion proteins was induced for 24 h with 5 mM Cu 2 S0 4 and verified by Western blotting of cell extracts using antibodies against β-gal (Cappel from MP Biomedicals, Irvine, California, United States). To assess β-gal activity, cells were rinsed in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min, rinsed twice in PBS, and incubated in staining solution [5 mM K 3 Fe(CN) 6, 5 mM K 4 Fe(CN) 6.6H 2 0, 2 mM MgCl 2, 1 mg/ml X-gal, in PBS] for 30 min at 37 °C. After staining, cells were rinsed in PBS and viewed on an inverted microscope.
Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing with PBS, the sections were incubated with goat-α-rabbit conjugated to 5 nm gold particles (Amersham Biosciences, Little Chalfont, United Kingdom). Incubations were stopped by buffer washes followed by fixation with 2.5% glutaraldehyde. Sections were further processed as described above except embedding was in EPON. Silver sections were collected on copper grids and viewed unstained in a JEOL 100 CX electron microscope operated at 80 kV. Distributions of gold particles were scored blindly by an impartial observer.
Machine-readable layer
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"text": "(C) Salivary glands expressing GFP:OR83b ( AB1-Gal4/+;UAS-GFP:Or83b/+ ) were stained with antibodies against the epitopes, illustrated in red in the snake plots on the left, under permeabilized or unpermeabilized conditions. For clarity, only the red channel is shown. None of the antibodies stain control salivary glands under permeabilized conditions (unpublished data)."
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"text": "To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b neurons [ ]. This technique combines cell-type specific induction of a UAS-Or83b transgene by an Or22a-Gal4 driver line with temporal regulation through use of a ubiquitously expressed temperature-sensitive Gal80 (Gal80ts) transgene. At the permissive temperature (18 °C), the GAL80 protein is active and represses induction of OR83b expression by inhibiting GAL4 ( A, top row). At the restrictive temperature (29 °C), GAL80 is inactivated, permitting expression of OR83b in OR22a/b neurons ( A, bottom row)."
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"text": "Antennae: 14-µm frozen sections of antennae were collected and stained with primary and secondary antibodies (see below) as described [ ]. Salivary glands: the anterior quarter of the third instar larvae was separated from the rest of the body, inverted to expose the salivary glands, and fixed in 4% formaldehyde in PBS for 20 min. These were then stained as for antennal sections, either in the presence of 0.25% Triton X-100 (Fisher Scientific, Springfield, New Jersey, United States) (permeabilized) or without detergent (non-permeabilized). After mounting in Vectashield (Vector Labs, Burlingame, California, United States), salivary glands were dissected away from other tissues. Images were collected with a Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany)."
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"text": "In vivo preparation of flies (3- to 8-d-old animals) and optical imaging of odor-evoked calcium responses in the antennal lobe were essentially as described [ ] using a modified TILL Photonics imaging system (TILL Photonics, Ludwig Maximilians University, Munich, Germany). For each measurement, a series of 40 frames was taken at 4 Hz. A constant air stream (2,000 ml/min) produced by an aquarium pump was guided through a 1-ml syringe with the tip placed 1 cm from the antennae. Pure odorants were diluted in paraffin oil (see figure legends for concentrations), and 10 µl of this solution was placed on a filter paper in a second syringe that was laterally inserted into this air flow. Odorant stimuli (1 s, i.e., frames 12-16) were puffed into the constant airstream using a custom-made electronic valve under computer control with a 1-min interstimulus interval. Imaging data were..."
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"text": "lacZ and TM:lacZ (encoding β-gal with an upstream synthetic TM domain) were amplified from pPD16.43 and pPD34.110 [ ], respectively, and cloned downstream of OR and Rh1 cDNA fragments in pMT-V5-His A (Invitrogen). Codons of test genes fused to lacZ and TM:lacZ: OR83b (1-46, 1-72), OR9a (1-41, 1-68), RH1 (1-49, 1-82). Plasmids were transfected into S2-R+ cultured Drosophila cells [ ] with Fugene (Qiagen, Valencia, California, United States). Expression of fusion proteins was induced for 24 h with 5 mM Cu 2 S0 4 and verified by Western blotting of cell extracts using antibodies against β-gal (Cappel from MP Biomedicals, Irvine, California, United States). To assess β-gal activity, cells were rinsed in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 5 min, rinsed twice in PBS, and incubated in staining solution [5 mM K 3 Fe(..."
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"text": "Prelabeling immunoelectron microscopy: 6-µm cryostat sections of fresh-frozen adult antennae were collected on an Aclar film (EMS) treated with Alcian blue. Sections were fixed for 10 min in 4% paraformaldehyde, washed in PBS, and incubated with α-EC2 (diluted 1:5,000) overnight in the cold. After washing with PBS, the sections were incubated with goat-α-rabbit conjugated to 5 nm gold particles (Amersham Biosciences, Little Chalfont, United Kingdom). Incubations were stopped by buffer washes followed by fixation with 2.5% glutaraldehyde. Sections were further processed as described above except embedding was in EPON. Silver sections were collected on copper grids and viewed unstained in a JEOL 100 CX electron microscope operated at 80 kV. Distributions of gold particles were scored blindly by an impartial observer."
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"name": "ORs and OR83b Form Heteromeric Complexes in the Sensory Cilia of OSNs"
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"headline": "Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo",
"datePublished": "2006",
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