BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth methods
Aim. Evidence-backed execution summary for BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth methods from BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.
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Subject model for the experiment.
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- confirm full cohort details in the source paper
BRs promote accumulation of a faster-migrating form of the PIF4 factor
reagent used in the protocol.
- Use
- DELLAs repress growth by binding the bHLH DNA recognition domain of PIFs, which blocks the DNA-binding ability of these factors (; ). The finding that BL promotes growth of mutants overaccumulating DELLAs and that these repressors are destabilized in det2-1 plants suggested that BRs may act at the level of PIFs. To...
PIF4-OE lines show a hyposensitive response to BRZ and activated levels of expression of BR-regulated genes
reagent used in the protocol.
- Use
- If BRs act through the PIFs, higher levels of PIF4 activity should phenocopy BL effects. Indeed, PIF4 -OE seedlings grown on BRZ are taller than wild-type controls, and these plants show a saturated response to BL (Supplemental Fig. S4). pif4pif5 seedlings, in contrast, display a hyposensitive response to this hormo...
PIF4 interacts with the BR signaling kinase BIN2
reagent used in the protocol.
- Use
- PIF4 interacts with the BR pathway kinase BIN2 in vitro and in vivo. ( A ) Yeast two-hybrid assay showing PIF4 and BIN2 interaction. Bait (BD) and prey (AD) constructs were cotransformed into yeast cells as indicated. Growth of yeast cells on SD-His Ade medium indicates interaction. The BIN2-BD and BES1-AD interacti...
PIF4 interacts with the BR signaling kinase BIN2
reagent used in the protocol.
- Use
- BIN2 phosphorylates PIF4 in in vitro kinase assays. The PIF4 and BIN2 interaction is mediated by the bHLH PIF4 domain and residues immediately adjacent to this domain. ( A ) BIN2 phosphorylates PIF4 in vitro. PIF4-6xHis, PIF41A-6xHis, and MBP-BES1 were incubated alone or with the BIN2 kinase fused to GST and 32 P-&#...
BIN2 phosphorylates PIF4 in vitro
reagent used in the protocol.
- Use
- In vitro kinase assays using the purified GST-BIN2, PIF4-6xHis, and MBP-BES1 proteins showed that BIN2 is able to phosphorylate both BES1 and PIF4 ( ). Labeling of these factors is only observed when BIN2 is added to the reaction mix. Also, although in pull-down assays BIN2 bound with similar affinities the BES1 and...
BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
reagent used in the protocol.
- Use
- BIN2 modulates PIF4 stability at dawn. ( A ) Phenotype of pif4pif5 and transgenic pif4pif5 lines expressing the wild-type PIF4-HA and mutant PIF41A-HA proteins under the control of its native promoter. pPIF4 ∷PIF41A-HA pif4pif5 lines show elongated petioles and narrow leaves, characteristic of plants grown in...
BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
reagent used in the protocol.
- Use
- Mutation of the conserved BIN2 phosphorylation motif increases the half-life of the PIF41A protein and leads to continuous growth. ( A ) Analysis of the half-life of the PIF4 and PIF41A proteins. Transgenic pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 seedlings were grown in SD for 5 d and treat...
PIF41A bes1-D seedlings are insensitive to BRZ in the light
reagent used in the protocol.
- Use
- The gain-of-function bes1-D and bzr1-1D mutations cause constitutive BES1 and BZR1 activation due to identical mutations in the PEST domain, which enhance PP2A-binding affinity and lead to increased dephosphorylation of these factors ( ). These mutants show a BRZ-insensitive response in the dark but opposite phenoty...
Materials and methods
Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumul...
- Use
- Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumul...
Protein interaction assays
The full-length BIN2, PIF4, and BES1 coding regions were cloned into the pENTRY S/D-TOPO vector for recombination into the yeast and plant expression vectors. Yeast two-hybrid assays were performed with the GATEWAY-modified pGBKT7 and pGADT7 vectors (Clontech). Both bait and prey constructs were transformed into AH1...
- Use
- The full-length BIN2, PIF4, and BES1 coding regions were cloned into the pENTRY S/D-TOPO vector for recombination into the yeast and plant expression vectors. Yeast two-hybrid assays were performed with the GATEWAY-modified pGBKT7 and pGADT7 vectors (Clontech). Both bait and prey constructs were transformed into AH1...
qRT-PCR gene expression analysis
Total RNA was extracted by using the high pure RNA isolation kit (Roche). The SuperScript II reverse transcriptase (Invitrogen) was used for cDNA synthesis, and quantitative real-time RT-PCR amplification wasperformed in the 7500 real-time PCR system (Applied Biosystems), following the manufacture's reco...
- Use
- Total RNA was extracted by using the high pure RNA isolation kit (Roche). The SuperScript II reverse transcriptase (Invitrogen) was used for cDNA synthesis, and quantitative real-time RT-PCR amplification wasperformed in the 7500 real-time PCR system (Applied Biosystems), following the manufacture's reco...
Confocal microscopy
GFP fluorescence of the split YFP in BiFC-infiltrated N. benthamiana leaves or in roots of the transgenic 35S ∷PIF4-GFP and 35S ∷PIF41A-GFP lines was imaged using an inverted Leica TCS SP5 spectral confocal microscope. Fluorescence was excited with a 488-nm ion argon laser, and emission images were colle...
- Use
- GFP fluorescence of the split YFP in BiFC-infiltrated N. benthamiana leaves or in roots of the transgenic 35S ∷PIF4-GFP and 35S ∷PIF41A-GFP lines was imaged using an inverted Leica TCS SP5 spectral confocal microscope. Fluorescence was excited with a 488-nm ion argon laser, and emission images were colle...
BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
Software used for acquisition, scoring, statistics, or reporting.
- Use
- BIN2 modulates PIF4 stability at dawn. ( A ) Phenotype of pif4pif5 and transgenic pif4pif5 lines expressing the wild-type PIF4-HA and mutant PIF41A-HA proteins under the control of its native promoter. pPIF4 ∷PIF41A-HA pif4pif5 lines show elongated petioles and narrow leaves, characteristic of plants grown in...
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BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
To estimate the stabilization of this mutant protein, we treated PIF4 and PIF41A seedlings with cycloheximide (CHX) and with CHX and BL ( ). In these studies, PIF4 is destabilized by 30 min of CHX application, while PIF41A is still detectable after 90 min of CHX treatment. Also, upon BL application, PIF4 half-life was similar to that of the PIF41A protein, whereas this hormone did not have any effect on PIF41A protein levels ( ). Consistent with these results, when PIF4 and PIF41A lines were crossed to the bin2.1 mutant, we observed that constitutive activation of BIN2 in these seedlings leads to a strong reduction in PIF4 protein levels but has only a minor effect on PIF41A protein stability ( ).
BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
Mutation of the conserved BIN2 phosphorylation motif increases the half-life of the PIF41A protein and leads to continuous growth. ( A ) Analysis of the half-life of the PIF4 and PIF41A proteins. Transgenic pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 seedlings were grown in SD for 5 d and treated for 12 h with 0.5 µM BRZ before starting the assay. Three hours before lights on, seedlings were changed to 0.5 µM BRZ (mock) or 1 µM BL (BL) medium, and 50 µM CHX was added 1 h later. Seedlings were kept in darkness and harvested at different times after CHX application, as indicated. Western blot analyses of these samples showed that the wild-type PIF4 protein is destabilized after 30 min of CHX treatment, in contrast to PIF41A, which is still detected by 90 min of CHX application. BL treatment increased PIF4 stability but did not have any effect on...
The concerted action of PIFs and BES1/BZR1 modulates rhythmic hypocotyl growth
Although it is widely accepted that PIFs accumulate only in darkness, we show that the PIF4 protein follows a pattern of accumulation similar to that of its transcript. We generated pif4pif5 lines expressing the PIF4-HA or PIF41A-HA proteins under the control of its native promoter and selected lines with wild-type levels of these transgenes. pPIF4 ∷PIF4-HA lines had a hypocotyl length and expressed the PIL1 and PRE5 genes to levels similar to those of the Col-0 plants (; Supplemental Figs. S7, S12). pPIF4 ∷PIF41A-HA lines, in contrast, were much taller and showed increased levels of expression of these genes ( ). pPIF4 ∷PIF41A plants are actually taller than the bes1-D mutant and, in the presence of BRZ, show increased levels of expression of the PIL1 and PRE5 transcripts, suggesting that PIF4 destabilization plays an important role in BRZ repression of hypocotyl g...
Materials and methods
Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark).
In vitro kinase and phosphorylation assays
PIF4-His, PIF41A-His, MBP-BES1, and BIN2-GST fusion proteins were purified using glutathione agarose (Clontech), amylose agarose (New England Biolabs), or Ni-NTA agarose (Qiagen) beads. For in vitro kinase assays, PIF4-His, MBP-BES1, and GST-BIN2 proteins were coincubated with 32 Pγ-ATP for 40 min at 37°C. Two micromolar and 20 µM of the BIN2 kinase inhibitor bikinin (Calbiochem) were added to the incubation mix for specific inhibition. Extracts of mock-, BL-, and BRZ-treated seedlings were incubated for 1 h at 37°C with or without CIP (New England Biolabs) to assay for in vivo protein phosphorylation.
Measurement outputs
What raw and processed outputs should exist?
If BRs act through the PIFs, higher levels of PIF4 activity should phenocopy BL effects. Indeed, PIF4 -OE seedlings grown on BRZ are taller than wild-type controls, and these pl...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
In line with such a BR-related phenotype, a significant overlap is observed between the genes differentially expressed in PIF4 -OE lines grown in red light ( ) and in dark-grown...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The GSK3-like kinase BIN2 negatively regulates BR signaling by inhibiting BES1/BZR1 transcriptional activity. BIN2-mediated phosphorylation of these factors inhibits DNA binding...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
PIF41A overexpression causes a severely elongated phenotype and activated PIL1 gene expression in the presence of BRZ. ( A ) Overexpression of the PIF41A mutant protein causes s...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
In line with such a BR-related phenotype, a significant overlap is observed between the genes differentially expressed in PIF4 -OE lines grown in red light ( ) and in dark-grown pifq seedlings (for dark data set, see; for pifq data set, see ) and those induced by BL treatment...
from paperScoring or quantification
Quantify the primary readouts for this experiment: If BRs act through the PIFs, higher levels of PIF4 activity should phenocopy BL effects. Indeed, PIF4 -OE seedlings grown on BRZ are taller than wild-type controls, and these pl...; In line with such a BR-related phenotype, a significant overlap is observed between the genes differentially expressed in PIF4 -OE lines grown in red light ( ) and in dark-grown...; The GSK3-like kinase BIN2 negatively regulates BR signaling by inhibiting BES1/BZR1 transcriptional activity. BIN2-mediated phosphorylation of these factors inhibits DNA binding...; PIF41A overexpression causes a severely elongated phenotype and activated PIL1 gene expression in the presence of BRZ. ( A ) Overexpression of the PIF41A mutant protein causes s....
from paperStatistical comparison
In line with such a BR-related phenotype, a significant overlap is observed between the genes differentially expressed in PIF4 -OE lines grown in red light ( ) and in dark-grown...; BIN2 modulates PIF4 stability at dawn. ( A ) Phenotype of pif4pif5 and transgenic pif4pif5 lines expressing the wild-type PIF4-HA and mutant PIF41A-HA proteins under the control...; Notably, although PIFs are accepted to be destabilized by PHYB in the light, both PIF4 and PIF41A proteins were detected to significant levels during daytime and rapidly dropped...; PIF41A bes1-d seedlings are insensitive to BRZ in the light. ( A ) Seedlings were grown for 5 d in continuous red light in a medium containing mock solution ( top panel) or 2 &#...
from paperReporting output
Report representative outputs alongside summary comparisons for If BRs act through the PIFs, higher levels of PIF4 activity should phenocopy BL effects. Indeed, PIF4 -OE seedlings grown on BRZ are taller than wild-type controls, and these pl..., In line with such a BR-related phenotype, a significant overlap is observed between the genes differentially expressed in PIF4 -OE lines grown in red light ( ) and in dark-grown..., The GSK3-like kinase BIN2 negatively regulates BR signaling by inhibiting BES1/BZR1 transcriptional activity. BIN2-mediated phosphorylation of these factors inhibits DNA binding..., PIF41A overexpression causes a severely elongated phenotype and activated PIL1 gene expression in the presence of BRZ. ( A ) Overexpression of the PIF41A mutant protein causes s....
inferred from protocolStructured statistical methods
In line with such a BR-related phenotype, a significant overlap is observed between the genes differentially expressed in PIF4 -OE lines grown in red light ( ) and in dark-grown...; BIN2 modulates PIF4 stability at dawn. ( A ) Phenotype of pif4pif5 and transgenic pif4pif5 lines expressing the wild-type PIF4-HA and mutant PIF41A-HA proteins under the control...; Notably, although PIFs are accepted to be destabilized by PHYB in the light, both PIF4 and PIF41A proteins were detected to significant levels during daytime and rapidly dropped...; PIF41A bes1-d seedlings are insensitive to BRZ in the light. ( A ) Seedlings were grown for 5 d in continuous red light in a medium containing mock solution ( top panel) or 2 &#...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
To estimate the stabilization of this mutant protein, we treated PIF4 and PIF41A seedlings with cycloheximide (CHX) and with CHX and BL ( ). In these studies, PIF4 is destabilized by 30 min of CHX application, while PIF41A is still detectable after 90 min of CHX treatment. Also, upon BL application, PIF4 half-life was similar to that of the PIF41A protein, whereas this hormone did not have any effect on PIF41A protein levels ( ). Consistent with these results, when PIF4 and PIF41A lines were crossed to the bin2.1 mutant, we observed that constitutive activation of BIN2 in these seedlings leads to a strong reduction in PIF4 protein levels but has only a minor effect on PIF41A protein stability ( ).
Mutation of the conserved BIN2 phosphorylation motif increases the half-life of the PIF41A protein and leads to continuous growth. ( A ) Analysis of the half-life of the PIF4 and PIF41A proteins. Transgenic pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 seedlings were grown in SD for 5 d and treated for 12 h with 0.5 µM BRZ before starting the assay. Three hours before lights on, seedlings were changed to 0.5 µM BRZ (mock) or 1 µM BL (BL) medium, and 50 µM CHX was added 1 h later. Seedlings were kept in darkness and harvested at different times after CHX application, as indicated. Western blot analyses of these samples showed that the wild-type PIF4 protein is destabilized after 30 min of CHX treatment, in contrast to PIF41A, which is still detected by 90 min of CHX application. BL treatment increased PIF4 stability but did not have any effect on the PIF1A protein. Note that in BL-treated seedlings, the half-life of PIF4 becomes similar to that of PIF41A. ( B ) Stability of the PIF4 and PIF41A protein in the bin2.1 mutant background. pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 lines were crossed with the bin2.1 mutant,...
Although it is widely accepted that PIFs accumulate only in darkness, we show that the PIF4 protein follows a pattern of accumulation similar to that of its transcript. We generated pif4pif5 lines expressing the PIF4-HA or PIF41A-HA proteins under the control of its native promoter and selected lines with wild-type levels of these transgenes. pPIF4 ∷PIF4-HA lines had a hypocotyl length and expressed the PIL1 and PRE5 genes to levels similar to those of the Col-0 plants (; Supplemental Figs. S7, S12). pPIF4 ∷PIF41A-HA lines, in contrast, were much taller and showed increased levels of expression of these genes ( ). pPIF4 ∷PIF41A plants are actually taller than the bes1-D mutant and, in the presence of BRZ, show increased levels of expression of the PIL1 and PRE5 transcripts, suggesting that PIF4 destabilization plays an important role in BRZ repression of hypocotyl growth. Time-course studies to analyze diurnal levels of accumulation of the PIF4 and PIF41A proteins showed that the wild-type PIF4 protein starts to accumulate at the end of the night, peaks during the day, and is reduced after transition to dark. PIF41A protein levels were higher than those of the...
Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark).
PIF4-His, PIF41A-His, MBP-BES1, and BIN2-GST fusion proteins were purified using glutathione agarose (Clontech), amylose agarose (New England Biolabs), or Ni-NTA agarose (Qiagen) beads. For in vitro kinase assays, PIF4-His, MBP-BES1, and GST-BIN2 proteins were coincubated with 32 Pγ-ATP for 40 min at 37°C. Two micromolar and 20 µM of the BIN2 kinase inhibitor bikinin (Calbiochem) were added to the incubation mix for specific inhibition. Extracts of mock-, BL-, and BRZ-treated seedlings were incubated for 1 h at 37°C with or without CIP (New England Biolabs) to assay for in vivo protein phosphorylation.
Machine-readable layer
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"name": "BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth methods",
"description": "Evidence-backed execution summary for BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth methods from BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth.",
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"text": "To estimate the stabilization of this mutant protein, we treated PIF4 and PIF41A seedlings with cycloheximide (CHX) and with CHX and BL ( ). In these studies, PIF4 is destabilized by 30 min of CHX application, while PIF41A is still detectable after 90 min of CHX treatment. Also, upon BL application, PIF4 half-life was similar to that of the PIF41A protein, whereas this hormone did not have any effect on PIF41A protein levels ( ). Consistent with these results, when PIF4 and PIF41A lines were crossed to the bin2.1 mutant, we observed that constitutive activation of BIN2 in these seedlings leads to a strong reduction in PIF4 protein levels but has only a minor effect on PIF41A protein stability ( )."
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"name": "BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn",
"text": "Mutation of the conserved BIN2 phosphorylation motif increases the half-life of the PIF41A protein and leads to continuous growth. ( A ) Analysis of the half-life of the PIF4 and PIF41A proteins. Transgenic pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 seedlings were grown in SD for 5 d and treated for 12 h with 0.5 µM BRZ before starting the assay. Three hours before lights on, seedlings were changed to 0.5 µM BRZ (mock) or 1 µM BL (BL) medium, and 50 µM CHX was added 1 h later. Seedlings were kept in darkness and harvested at different times after CHX application, as indicated. Western blot analyses of these samples showed that the wild-type PIF4 protein is destabilized after 30 min of CHX treatment, in contrast to PIF41A, which is still detected by 90 min of CHX application. BL treatment increased PIF4 stability but did not have any effect on..."
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"text": "Although it is widely accepted that PIFs accumulate only in darkness, we show that the PIF4 protein follows a pattern of accumulation similar to that of its transcript. We generated pif4pif5 lines expressing the PIF4-HA or PIF41A-HA proteins under the control of its native promoter and selected lines with wild-type levels of these transgenes. pPIF4 ∷PIF4-HA lines had a hypocotyl length and expressed the PIL1 and PRE5 genes to levels similar to those of the Col-0 plants (; Supplemental Figs. S7, S12). pPIF4 ∷PIF41A-HA lines, in contrast, were much taller and showed increased levels of expression of these genes ( ). pPIF4 ∷PIF41A plants are actually taller than the bes1-D mutant and, in the presence of BRZ, show increased levels of expression of the PIL1 and PRE5 transcripts, suggesting that PIF4 destabilization plays an important role in BRZ repression of hypocotyl g..."
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"text": "Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark)."
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"text": "PIF4-His, PIF41A-His, MBP-BES1, and BIN2-GST fusion proteins were purified using glutathione agarose (Clontech), amylose agarose (New England Biolabs), or Ni-NTA agarose (Qiagen) beads. For in vitro kinase assays, PIF4-His, MBP-BES1, and GST-BIN2 proteins were coincubated with 32 Pγ-ATP for 40 min at 37°C. Two micromolar and 20 µM of the BIN2 kinase inhibitor bikinin (Calbiochem) were added to the incubation mix for specific inhibition. Extracts of mock-, BL-, and BRZ-treated seedlings were incubated for 1 h at 37°C with or without CIP (New England Biolabs) to assay for in vivo protein phosphorylation."
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"name": "PIF4 interacts with the BR signaling kinase BIN2"
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"name": "BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn"
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"datePublished": "2014",
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"identifier": "10.1101/gad.243675.114"
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