Structured Review

Becton Dickinson anti fak
<t>PKL</t> is tyrosine-phosphorylated by Src and <t>FAK</t> in response to PDGF. (A) Endogenous PKL and GIT1 were precipitated from quiescent and PDGF (20 ng/ml)-stimulated MEFs and blotted with phosphotyrosine antibody (PY, clone 4G10). (B) MEFs were transiently transfected
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1) Product Images from "Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration"

Article Title: Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E09-07-0548

PKL is tyrosine-phosphorylated by Src and FAK in response to PDGF. (A) Endogenous PKL and GIT1 were precipitated from quiescent and PDGF (20 ng/ml)-stimulated MEFs and blotted with phosphotyrosine antibody (PY, clone 4G10). (B) MEFs were transiently transfected
Figure Legend Snippet: PKL is tyrosine-phosphorylated by Src and FAK in response to PDGF. (A) Endogenous PKL and GIT1 were precipitated from quiescent and PDGF (20 ng/ml)-stimulated MEFs and blotted with phosphotyrosine antibody (PY, clone 4G10). (B) MEFs were transiently transfected

Techniques Used: Transfection

2) Product Images from "Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration"

Article Title: Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E09-07-0548

PKL and its tyrosine phosphorylation regulate PAK activity. (A and B) Control or PKL RNAi MEFs were stimulated with PDGF and blotted with PAK pS199/204 and pan PAK (N-20) antibodies. PKL RNAi knockdown was confirmed by blotting for PKL. Quantification
Figure Legend Snippet: PKL and its tyrosine phosphorylation regulate PAK activity. (A and B) Control or PKL RNAi MEFs were stimulated with PDGF and blotted with PAK pS199/204 and pan PAK (N-20) antibodies. PKL RNAi knockdown was confirmed by blotting for PKL. Quantification

Techniques Used: Activity Assay

PKL/GIT2 is required for directional cell migration and cell polarity. (A) PKL RNAi knockdown in MEFs. Normal MEFs were transfected with mouse-specific siRNA for PKL. At 60 h after transfection, cells were lysed and subjected to Western immunoblotting.
Figure Legend Snippet: PKL/GIT2 is required for directional cell migration and cell polarity. (A) PKL RNAi knockdown in MEFs. Normal MEFs were transfected with mouse-specific siRNA for PKL. At 60 h after transfection, cells were lysed and subjected to Western immunoblotting.

Techniques Used: Migration, Transfection, Western Blot

PKL and PKL tyrosine phosphorylation regulates phospho-Erk signaling. (A) Serum starved control RNAi and PKL RNAi cells were stimulated with 20 ng/ml PDGF (5, 10, 30, and 60 min) followed by lysis. Lysates were blotted with phospho-Erk, pan-Erk, and PKL
Figure Legend Snippet: PKL and PKL tyrosine phosphorylation regulates phospho-Erk signaling. (A) Serum starved control RNAi and PKL RNAi cells were stimulated with 20 ng/ml PDGF (5, 10, 30, and 60 min) followed by lysis. Lysates were blotted with phospho-Erk, pan-Erk, and PKL

Techniques Used: Lysis

PKL and PKL tyrosine phosphorylation mediates Rac1 and Cdc42 activities. (A and B) Control RNAi and PKL RNAi cells were cultured in serum-free medium for 4 h. Cells were stimulated with 20 ng/ml PDGF (10 min) followed by lysis. Rac1 activity was determined
Figure Legend Snippet: PKL and PKL tyrosine phosphorylation mediates Rac1 and Cdc42 activities. (A and B) Control RNAi and PKL RNAi cells were cultured in serum-free medium for 4 h. Cells were stimulated with 20 ng/ml PDGF (10 min) followed by lysis. Rac1 activity was determined

Techniques Used: Cell Culture, Lysis, Activity Assay

3) Product Images from "The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis"

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Journal: Plant Communications

doi: 10.1016/j.xplc.2019.100011

LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.
Figure Legend Snippet: LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.

Techniques Used: Chromatin Immunoprecipitation

PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.
Figure Legend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.
Figure Legend Snippet: LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.

Techniques Used: Expressing, Sequencing

4) Product Images from "Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration"

Article Title: Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E09-07-0548

PKL is tyrosine-phosphorylated by Src and FAK in response to PDGF. (A) Endogenous PKL and GIT1 were precipitated from quiescent and PDGF (20 ng/ml)-stimulated MEFs and blotted with phosphotyrosine antibody (PY, clone 4G10). (B) MEFs were transiently transfected
Figure Legend Snippet: PKL is tyrosine-phosphorylated by Src and FAK in response to PDGF. (A) Endogenous PKL and GIT1 were precipitated from quiescent and PDGF (20 ng/ml)-stimulated MEFs and blotted with phosphotyrosine antibody (PY, clone 4G10). (B) MEFs were transiently transfected

Techniques Used: Transfection

5) Product Images from "The Cell Adhesion-associated Protein Git2 Regulates Morphogenetic Movements during Zebrafish Embryonic Development"

Article Title: The Cell Adhesion-associated Protein Git2 Regulates Morphogenetic Movements during Zebrafish Embryonic Development

Journal: Developmental biology

doi: 10.1016/j.ydbio.2010.10.027

Identification and characterization of git2 genes in zebrafish ( A ) Phylogenetic analysis of zebrafish git2 family genes. Dendogram of zebrafish git2a on chromosome 5 and git2b on chromosome 10 and related orthologs from other species. ( B ) in situ hybridization of git2a mRNA expression in the zebrafish embryo. git2a expression was ubiquitously detected at the 4-cell, epiboly, tailbud and 14-somite (14SS) and 24hpf stages. ( C ) Western blotting of zebrafish Git2 protein at dome, 50%, 75%, 90% epiboly and 6-somite (6SS) stages, α-Tubulin and paxillin were used as loading controls. ( D ) Immunohistochemistry of Git2 (red) at the 75% epiboly stage. Embryos were co-stained with phalloidin to detect F-actin (green). Images show surface EVL cells and deep cells (30μm below the surface). Scale bar, 50μm. Fluorescent intensity profiles show relative F-actin (green) and Git2 (red) levels in EVL cells at the blastoderm margin (1) and deep cells (2). ( E .
Figure Legend Snippet: Identification and characterization of git2 genes in zebrafish ( A ) Phylogenetic analysis of zebrafish git2 family genes. Dendogram of zebrafish git2a on chromosome 5 and git2b on chromosome 10 and related orthologs from other species. ( B ) in situ hybridization of git2a mRNA expression in the zebrafish embryo. git2a expression was ubiquitously detected at the 4-cell, epiboly, tailbud and 14-somite (14SS) and 24hpf stages. ( C ) Western blotting of zebrafish Git2 protein at dome, 50%, 75%, 90% epiboly and 6-somite (6SS) stages, α-Tubulin and paxillin were used as loading controls. ( D ) Immunohistochemistry of Git2 (red) at the 75% epiboly stage. Embryos were co-stained with phalloidin to detect F-actin (green). Images show surface EVL cells and deep cells (30μm below the surface). Scale bar, 50μm. Fluorescent intensity profiles show relative F-actin (green) and Git2 (red) levels in EVL cells at the blastoderm margin (1) and deep cells (2). ( E .

Techniques Used: In Situ Hybridization, Expressing, Western Blot, Immunohistochemistry, Staining

Embryonic phenotypes following Git2a morpholino knockdown ( A ) Live embryos injected with either control morpholino (MO) or git2a MO at 24 hpf and 48 hpf. git2a morphants exhibited variable defects including a curled tail, a short body axis and edema. The graph shows the quantification of phenotypes at 24 hpf from three independent experiments. ( B ) Representative images of control and git2a morphant embryos at 6, 10 and 12 hpf. A delay or arrest of epiboly was observed in git2a morphants. ( C ) The percentage of control and git2a MO embryos showing epiboly defects. Co-injection of chicken GIT2 mRNA partially rescued these defects. Injection of git2a MO into the yolk cell resulted in only minor epiboly defects. ( D ) Timing of epiboly progression in uninjected, control MO, git2a MO injected and chicken GIT2 mRNA rescued embryos. Data combined from at least four independent experiments. ( E ) Representative images of chicken GIT2 mRNA rescue phenotypes compared with control and git2a morphants at 9 and 24 hpf. ( F ) Western blot for Git2 protein expression in git2a morphants and embryos co-injected with git2a MO and chicken GIT2 mRNA.
Figure Legend Snippet: Embryonic phenotypes following Git2a morpholino knockdown ( A ) Live embryos injected with either control morpholino (MO) or git2a MO at 24 hpf and 48 hpf. git2a morphants exhibited variable defects including a curled tail, a short body axis and edema. The graph shows the quantification of phenotypes at 24 hpf from three independent experiments. ( B ) Representative images of control and git2a morphant embryos at 6, 10 and 12 hpf. A delay or arrest of epiboly was observed in git2a morphants. ( C ) The percentage of control and git2a MO embryos showing epiboly defects. Co-injection of chicken GIT2 mRNA partially rescued these defects. Injection of git2a MO into the yolk cell resulted in only minor epiboly defects. ( D ) Timing of epiboly progression in uninjected, control MO, git2a MO injected and chicken GIT2 mRNA rescued embryos. Data combined from at least four independent experiments. ( E ) Representative images of chicken GIT2 mRNA rescue phenotypes compared with control and git2a morphants at 9 and 24 hpf. ( F ) Western blot for Git2 protein expression in git2a morphants and embryos co-injected with git2a MO and chicken GIT2 mRNA.

Techniques Used: Injection, Western Blot, Expressing

Cell morphology is disrupted by Git2a knockdown Representative images of cortical actin and Git2 distribution in EVL cells of control and git2a morphant embryos at 75% epiboly. Git2 expression was reduced and cell morphology, outlined by cortical actin, was significantly altered in git2a ).
Figure Legend Snippet: Cell morphology is disrupted by Git2a knockdown Representative images of cortical actin and Git2 distribution in EVL cells of control and git2a morphant embryos at 75% epiboly. Git2 expression was reduced and cell morphology, outlined by cortical actin, was significantly altered in git2a ).

Techniques Used: Expressing

Git2 functions through myosin II-dependent contractility to regulate epiboly ( A ) Immunofluoresence staining of phosphorylated Myosin light chain (pMLC) and F-actin in EVL cells of control and git2a morphants at 75% epiboly. In control embryos, pMLC (red) co-localized with cortical actin (green) at the cell periphery in EVL cells and at the margin where the EVL contacts the YSL. However, a significant reduction of pMLC staining was observed in git2a morphants. Scale bar, 50μm. The F-actin and pMLC fluorescence pixel intensity profiles of EVL cells in control (1, 2) and git2a morphant (3, 4) embryos was aligned. ( B ) Western blotting of pMLC and total levels of MLC in control and git2a morphants from 30% epiboly to the 6 somite stage. ( C ) Quantification of the relative level of pMLC: total MLC from control and git2a morphants at 50% and 75% epiboly from three independent experiments. An arbitrary unit (AU) is designated as the pMLC level at 50% epiboly.
Figure Legend Snippet: Git2 functions through myosin II-dependent contractility to regulate epiboly ( A ) Immunofluoresence staining of phosphorylated Myosin light chain (pMLC) and F-actin in EVL cells of control and git2a morphants at 75% epiboly. In control embryos, pMLC (red) co-localized with cortical actin (green) at the cell periphery in EVL cells and at the margin where the EVL contacts the YSL. However, a significant reduction of pMLC staining was observed in git2a morphants. Scale bar, 50μm. The F-actin and pMLC fluorescence pixel intensity profiles of EVL cells in control (1, 2) and git2a morphant (3, 4) embryos was aligned. ( B ) Western blotting of pMLC and total levels of MLC in control and git2a morphants from 30% epiboly to the 6 somite stage. ( C ) Quantification of the relative level of pMLC: total MLC from control and git2a morphants at 50% and 75% epiboly from three independent experiments. An arbitrary unit (AU) is designated as the pMLC level at 50% epiboly.

Techniques Used: Staining, Fluorescence, Western Blot

6) Product Images from "Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility"

Article Title: Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020757

Binding of liprin-α1 to GIT1-C2 prevents binding of paxillin to GIT1-C2. (A) Lysates were prepared from COS7 cells transfected with either HA-GIT1-C2 (C2) or co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 (C2+Lip). Aliquots of the lysates were used for immunoprecipitation with anti-paxillin antibodies (IP anti-paxillin, 400 µg of protein per IP). Filters with immunoprecipitates (a), and with 100 µg of both lysates (Lys) and unbound fractions after IP (Ub) (b) were cut and immunoblotted with anti-Flag to detect Flag-liprin-α1 (upper filters, only one of the duplicated immunoprecipitations is shown); since GIT1-C2 and paxillin migrate at similar positions on gels, the lower parts of the filters from the duplicated immunoprecipitations were used as follows: one set of filters (a+b) was incubated with anti-HA to detect HA-GIT1-C2 (middle blots), and one set was incubated with anti-paxillin to detect endogenous paxillin (lower blots). Paxillin was absent from the unbound fractions after immunoprecipitation (Ub). (c) The unbound fraction (300 µg) after immunoprecipitation with anti-paxillin from the lysate of cells co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 [Ub(C2+Lip)], was re-immunoprecipitated with anti-liprin antibody, to reveal the presence of the liprin-α1/GIT1-C2 complex in the lysate. (B) Binding of liprin-α1 to GIT1-C2 does not prevent binding of βPIX to GIT1-C2. Identification of a ternary complex among liprin-α1, βPIX and GIT1-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-FLAG antibodies (top blots on the left). Aliquots of the unbound fraction after the first round of immunoprecipitations were re-immunoprecipitated with anti-βPIX antibodies (top blots on the right). Filters including immunoprecipitations (IP), lysates (Lys), and unbound fractions after the second round of immunoprecipitations (Ub) were cut and blotted as indicated (lower blots). (C) Liprin-α1 does not interfere with the interaction of βPIX with GIT-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-βPIX antibodies. Filters including aliquots of lysates and the immunoprecipitations (IP) were cut and blotted as indicated. (D) A COS7 cell lysate (1 mg protein) was immunoprecipitated with anti-βPIX antibodies. Immunoprecipitate (IP) and equal amounts (100 µg) of lysate (Lys) and unbound fraction (Ub) were blotted with anti-GIT (mAb PKL, recognizing both GIT1 and GIT2 proteins, on the left; or anti-GIT2-specific pAb, on the right), βPIX, or anti-liprin-α1 antibodies. Blot with anti-GIT antibody was performed after stripping the filter incubated for βPIX. (E) binding of βPIX to full length GIT1 does not enhance the binding of liprin-α1 to GIT1. COS7 cells were co-transfected with FLAG-liprin-α1 and FLAG-GIT1, or with FLAG-liprin-α1 and FLAG-GIT1 and HA-βPIX. 200 µg of each lysate were immunoprecipitated with anti-GIT1 antiserum. Lysates (Lys, 50 µg), unbound fractions (Ub, 50 µg) and immunoprecipitates were blotted and incubated with antibodies specific for the indicated proteins. Overexpression of βPix did not increase the interaction of liprin-α1 with GIT1. (F) Model for the regulated interaction of GIT1 with paxillin and liprin-α1. Either ligand binds poorly to full length GIT1. We hypothesize that activation of GIT1 by so far unknown mechanisms is required for the formation of either GIT1/paxillin or GIT1/liprin-α1 complexes.
Figure Legend Snippet: Binding of liprin-α1 to GIT1-C2 prevents binding of paxillin to GIT1-C2. (A) Lysates were prepared from COS7 cells transfected with either HA-GIT1-C2 (C2) or co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 (C2+Lip). Aliquots of the lysates were used for immunoprecipitation with anti-paxillin antibodies (IP anti-paxillin, 400 µg of protein per IP). Filters with immunoprecipitates (a), and with 100 µg of both lysates (Lys) and unbound fractions after IP (Ub) (b) were cut and immunoblotted with anti-Flag to detect Flag-liprin-α1 (upper filters, only one of the duplicated immunoprecipitations is shown); since GIT1-C2 and paxillin migrate at similar positions on gels, the lower parts of the filters from the duplicated immunoprecipitations were used as follows: one set of filters (a+b) was incubated with anti-HA to detect HA-GIT1-C2 (middle blots), and one set was incubated with anti-paxillin to detect endogenous paxillin (lower blots). Paxillin was absent from the unbound fractions after immunoprecipitation (Ub). (c) The unbound fraction (300 µg) after immunoprecipitation with anti-paxillin from the lysate of cells co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 [Ub(C2+Lip)], was re-immunoprecipitated with anti-liprin antibody, to reveal the presence of the liprin-α1/GIT1-C2 complex in the lysate. (B) Binding of liprin-α1 to GIT1-C2 does not prevent binding of βPIX to GIT1-C2. Identification of a ternary complex among liprin-α1, βPIX and GIT1-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-FLAG antibodies (top blots on the left). Aliquots of the unbound fraction after the first round of immunoprecipitations were re-immunoprecipitated with anti-βPIX antibodies (top blots on the right). Filters including immunoprecipitations (IP), lysates (Lys), and unbound fractions after the second round of immunoprecipitations (Ub) were cut and blotted as indicated (lower blots). (C) Liprin-α1 does not interfere with the interaction of βPIX with GIT-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-βPIX antibodies. Filters including aliquots of lysates and the immunoprecipitations (IP) were cut and blotted as indicated. (D) A COS7 cell lysate (1 mg protein) was immunoprecipitated with anti-βPIX antibodies. Immunoprecipitate (IP) and equal amounts (100 µg) of lysate (Lys) and unbound fraction (Ub) were blotted with anti-GIT (mAb PKL, recognizing both GIT1 and GIT2 proteins, on the left; or anti-GIT2-specific pAb, on the right), βPIX, or anti-liprin-α1 antibodies. Blot with anti-GIT antibody was performed after stripping the filter incubated for βPIX. (E) binding of βPIX to full length GIT1 does not enhance the binding of liprin-α1 to GIT1. COS7 cells were co-transfected with FLAG-liprin-α1 and FLAG-GIT1, or with FLAG-liprin-α1 and FLAG-GIT1 and HA-βPIX. 200 µg of each lysate were immunoprecipitated with anti-GIT1 antiserum. Lysates (Lys, 50 µg), unbound fractions (Ub, 50 µg) and immunoprecipitates were blotted and incubated with antibodies specific for the indicated proteins. Overexpression of βPix did not increase the interaction of liprin-α1 with GIT1. (F) Model for the regulated interaction of GIT1 with paxillin and liprin-α1. Either ligand binds poorly to full length GIT1. We hypothesize that activation of GIT1 by so far unknown mechanisms is required for the formation of either GIT1/paxillin or GIT1/liprin-α1 complexes.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Incubation, Stripping Membranes, Over Expression, Activation Assay

7) Product Images from "Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics"

Article Title: Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200608010

PYK2 and ILK still localize to FAs in Fak- null keratinocytes. Immunoblot analysis and immunofluorescence of WT and Fak KO MK (A) or WT and β1 KO MK (B). Antibodies used are as shown, except phalloidin (red) is used to mark F-actin, and DAPI (blue) labels nuclear chromatin. Phosphorylated versions of FAK and PYK2 are active. Antibodies are color coded according to the secondary antibodies used. Boxed areas are magnified and shown as insets in which phalloidin fluorescence has been omitted. Note that Fak KO MKs still display active PYK2 as well as FA-localized ILK.
Figure Legend Snippet: PYK2 and ILK still localize to FAs in Fak- null keratinocytes. Immunoblot analysis and immunofluorescence of WT and Fak KO MK (A) or WT and β1 KO MK (B). Antibodies used are as shown, except phalloidin (red) is used to mark F-actin, and DAPI (blue) labels nuclear chromatin. Phosphorylated versions of FAK and PYK2 are active. Antibodies are color coded according to the secondary antibodies used. Boxed areas are magnified and shown as insets in which phalloidin fluorescence has been omitted. Note that Fak KO MKs still display active PYK2 as well as FA-localized ILK.

Techniques Used: Immunofluorescence, Fluorescence

8) Product Images from "Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility"

Article Title: Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020757

GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression. (A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P
Figure Legend Snippet: GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression. (A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P

Techniques Used: Over Expression, Luciferase, Expressing, Transfection, SDS Page, Incubation, Cotransfection, Immunostaining

9) Product Images from "G-protein-coupled Receptor Kinase Interactor-1 (GIT1) Is a New Endothelial Nitric-oxide Synthase (eNOS) Interactor with Functional Effects on Vascular Homeostasis *"

Article Title: G-protein-coupled Receptor Kinase Interactor-1 (GIT1) Is a New Endothelial Nitric-oxide Synthase (eNOS) Interactor with Functional Effects on Vascular Homeostasis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.320465

GIT1 expression and its effect on eNOS activity in normal sinusoidal endothelial cells. A , sinusoidal endothelial cells were transfected with cDNA encoding full-length GIT1 ( GIT1 , 2 μg) or a cognate empty vector ( EV ). Phospho-eNOS (Ser 1177 ), total eNOS, and GIT1 were detected in cell lysates by immunoblotting, and representative images are shown. Nitrite was measured in conditioned medium from the same cells and is shown in the right graph ( n = 5, *, p
Figure Legend Snippet: GIT1 expression and its effect on eNOS activity in normal sinusoidal endothelial cells. A , sinusoidal endothelial cells were transfected with cDNA encoding full-length GIT1 ( GIT1 , 2 μg) or a cognate empty vector ( EV ). Phospho-eNOS (Ser 1177 ), total eNOS, and GIT1 were detected in cell lysates by immunoblotting, and representative images are shown. Nitrite was measured in conditioned medium from the same cells and is shown in the right graph ( n = 5, *, p

Techniques Used: Expressing, Activity Assay, Transfection, Plasmid Preparation

Overexpression of GIT1 in injured liver endothelial cells enhances NO production and ameliorates portal hypertension. A , sinusoidal endothelial cells isolated after BDL were transfected with GIT1 or EV, and cell lysates were subjected to immunoblotting ( IB ) with the indicated antibodies (representative immunoblots of 3 are shown). B , sinusoidal endothelial cells from BDL were transfected with GIT1 (0.5 to 1.5 μg) and nitrite levels from conditioned medium were measured ( n = 3, *, p
Figure Legend Snippet: Overexpression of GIT1 in injured liver endothelial cells enhances NO production and ameliorates portal hypertension. A , sinusoidal endothelial cells isolated after BDL were transfected with GIT1 or EV, and cell lysates were subjected to immunoblotting ( IB ) with the indicated antibodies (representative immunoblots of 3 are shown). B , sinusoidal endothelial cells from BDL were transfected with GIT1 (0.5 to 1.5 μg) and nitrite levels from conditioned medium were measured ( n = 3, *, p

Techniques Used: Over Expression, Isolation, Transfection, Western Blot

10) Product Images from "G-protein-coupled Receptor Kinase Interactor-1 (GIT1) Is a New Endothelial Nitric-oxide Synthase (eNOS) Interactor with Functional Effects on Vascular Homeostasis *"

Article Title: G-protein-coupled Receptor Kinase Interactor-1 (GIT1) Is a New Endothelial Nitric-oxide Synthase (eNOS) Interactor with Functional Effects on Vascular Homeostasis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.320465

GIT1 expression and its effect on eNOS activity in normal sinusoidal endothelial cells. A , sinusoidal endothelial cells were transfected with cDNA encoding full-length GIT1 ( GIT1 , 2 μg) or a cognate empty vector ( EV ). Phospho-eNOS (Ser 1177 ), total eNOS, and GIT1 were detected in cell lysates by immunoblotting, and representative images are shown. Nitrite was measured in conditioned medium from the same cells and is shown in the right graph ( n = 5, *, p
Figure Legend Snippet: GIT1 expression and its effect on eNOS activity in normal sinusoidal endothelial cells. A , sinusoidal endothelial cells were transfected with cDNA encoding full-length GIT1 ( GIT1 , 2 μg) or a cognate empty vector ( EV ). Phospho-eNOS (Ser 1177 ), total eNOS, and GIT1 were detected in cell lysates by immunoblotting, and representative images are shown. Nitrite was measured in conditioned medium from the same cells and is shown in the right graph ( n = 5, *, p

Techniques Used: Expressing, Activity Assay, Transfection, Plasmid Preparation

11) Product Images from "Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility"

Article Title: Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020757

Binding of liprin-α1 to GIT1-C2 prevents binding of paxillin to GIT1-C2. (A) Lysates were prepared from COS7 cells transfected with either HA-GIT1-C2 (C2) or co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 (C2+Lip). Aliquots of the lysates were used for immunoprecipitation with anti-paxillin antibodies (IP anti-paxillin, 400 µg of protein per IP). Filters with immunoprecipitates (a), and with 100 µg of both lysates (Lys) and unbound fractions after IP (Ub) (b) were cut and immunoblotted with anti-Flag to detect Flag-liprin-α1 (upper filters, only one of the duplicated immunoprecipitations is shown); since GIT1-C2 and paxillin migrate at similar positions on gels, the lower parts of the filters from the duplicated immunoprecipitations were used as follows: one set of filters (a+b) was incubated with anti-HA to detect HA-GIT1-C2 (middle blots), and one set was incubated with anti-paxillin to detect endogenous paxillin (lower blots). Paxillin was absent from the unbound fractions after immunoprecipitation (Ub). (c) The unbound fraction (300 µg) after immunoprecipitation with anti-paxillin from the lysate of cells co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 [Ub(C2+Lip)], was re-immunoprecipitated with anti-liprin antibody, to reveal the presence of the liprin-α1/GIT1-C2 complex in the lysate. (B) Binding of liprin-α1 to GIT1-C2 does not prevent binding of βPIX to GIT1-C2. Identification of a ternary complex among liprin-α1, βPIX and GIT1-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-FLAG antibodies (top blots on the left). Aliquots of the unbound fraction after the first round of immunoprecipitations were re-immunoprecipitated with anti-βPIX antibodies (top blots on the right). Filters including immunoprecipitations (IP), lysates (Lys), and unbound fractions after the second round of immunoprecipitations (Ub) were cut and blotted as indicated (lower blots). (C) Liprin-α1 does not interfere with the interaction of βPIX with GIT-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-βPIX antibodies. Filters including aliquots of lysates and the immunoprecipitations (IP) were cut and blotted as indicated. (D) A COS7 cell lysate (1 mg protein) was immunoprecipitated with anti-βPIX antibodies. Immunoprecipitate (IP) and equal amounts (100 µg) of lysate (Lys) and unbound fraction (Ub) were blotted with anti-GIT (mAb PKL, recognizing both GIT1 and GIT2 proteins, on the left; or anti-GIT2-specific pAb, on the right), βPIX, or anti-liprin-α1 antibodies. Blot with anti-GIT antibody was performed after stripping the filter incubated for βPIX. (E) binding of βPIX to full length GIT1 does not enhance the binding of liprin-α1 to GIT1. COS7 cells were co-transfected with FLAG-liprin-α1 and FLAG-GIT1, or with FLAG-liprin-α1 and FLAG-GIT1 and HA-βPIX. 200 µg of each lysate were immunoprecipitated with anti-GIT1 antiserum. Lysates (Lys, 50 µg), unbound fractions (Ub, 50 µg) and immunoprecipitates were blotted and incubated with antibodies specific for the indicated proteins. Overexpression of βPix did not increase the interaction of liprin-α1 with GIT1. (F) Model for the regulated interaction of GIT1 with paxillin and liprin-α1. Either ligand binds poorly to full length GIT1. We hypothesize that activation of GIT1 by so far unknown mechanisms is required for the formation of either GIT1/paxillin or GIT1/liprin-α1 complexes.
Figure Legend Snippet: Binding of liprin-α1 to GIT1-C2 prevents binding of paxillin to GIT1-C2. (A) Lysates were prepared from COS7 cells transfected with either HA-GIT1-C2 (C2) or co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 (C2+Lip). Aliquots of the lysates were used for immunoprecipitation with anti-paxillin antibodies (IP anti-paxillin, 400 µg of protein per IP). Filters with immunoprecipitates (a), and with 100 µg of both lysates (Lys) and unbound fractions after IP (Ub) (b) were cut and immunoblotted with anti-Flag to detect Flag-liprin-α1 (upper filters, only one of the duplicated immunoprecipitations is shown); since GIT1-C2 and paxillin migrate at similar positions on gels, the lower parts of the filters from the duplicated immunoprecipitations were used as follows: one set of filters (a+b) was incubated with anti-HA to detect HA-GIT1-C2 (middle blots), and one set was incubated with anti-paxillin to detect endogenous paxillin (lower blots). Paxillin was absent from the unbound fractions after immunoprecipitation (Ub). (c) The unbound fraction (300 µg) after immunoprecipitation with anti-paxillin from the lysate of cells co-transfected with HA-GIT1-C2 and FLAG-liprin-α1 [Ub(C2+Lip)], was re-immunoprecipitated with anti-liprin antibody, to reveal the presence of the liprin-α1/GIT1-C2 complex in the lysate. (B) Binding of liprin-α1 to GIT1-C2 does not prevent binding of βPIX to GIT1-C2. Identification of a ternary complex among liprin-α1, βPIX and GIT1-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-FLAG antibodies (top blots on the left). Aliquots of the unbound fraction after the first round of immunoprecipitations were re-immunoprecipitated with anti-βPIX antibodies (top blots on the right). Filters including immunoprecipitations (IP), lysates (Lys), and unbound fractions after the second round of immunoprecipitations (Ub) were cut and blotted as indicated (lower blots). (C) Liprin-α1 does not interfere with the interaction of βPIX with GIT-C2. COS7 cells co-transfected to express the indicated combinations of HA-GIT1-C2, HA-βPIX, and FLAG-liprin-α1 were immunoprecipitated with anti-βPIX antibodies. Filters including aliquots of lysates and the immunoprecipitations (IP) were cut and blotted as indicated. (D) A COS7 cell lysate (1 mg protein) was immunoprecipitated with anti-βPIX antibodies. Immunoprecipitate (IP) and equal amounts (100 µg) of lysate (Lys) and unbound fraction (Ub) were blotted with anti-GIT (mAb PKL, recognizing both GIT1 and GIT2 proteins, on the left; or anti-GIT2-specific pAb, on the right), βPIX, or anti-liprin-α1 antibodies. Blot with anti-GIT antibody was performed after stripping the filter incubated for βPIX. (E) binding of βPIX to full length GIT1 does not enhance the binding of liprin-α1 to GIT1. COS7 cells were co-transfected with FLAG-liprin-α1 and FLAG-GIT1, or with FLAG-liprin-α1 and FLAG-GIT1 and HA-βPIX. 200 µg of each lysate were immunoprecipitated with anti-GIT1 antiserum. Lysates (Lys, 50 µg), unbound fractions (Ub, 50 µg) and immunoprecipitates were blotted and incubated with antibodies specific for the indicated proteins. Overexpression of βPix did not increase the interaction of liprin-α1 with GIT1. (F) Model for the regulated interaction of GIT1 with paxillin and liprin-α1. Either ligand binds poorly to full length GIT1. We hypothesize that activation of GIT1 by so far unknown mechanisms is required for the formation of either GIT1/paxillin or GIT1/liprin-α1 complexes.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Incubation, Stripping Membranes, Over Expression, Activation Assay

GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression. (A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P
Figure Legend Snippet: GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression. (A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P

Techniques Used: Over Expression, Luciferase, Expressing, Transfection, SDS Page, Incubation, Cotransfection, Immunostaining

Expression of GIT1-C affects cell morphology and the distribution of endogenous liprin-α1. (A) COS7 cells transfected for one day with either FLAG-GIT1, FLAG-GIT1-C, or FLAG-βGalactosidase were re-plated for 1 h on FN. Immunofluorescence for the transfected proteins (FLAG), paxillin, and phalloidin staining for F-actin. Scale bar, 20 µm. Below, 3-fold enlargements of areas from cells stained for paxillin (arrowheads in the corresponding cells above) are shown. (B) Expression of GIT1-C induces a significant increase of cell spreading on FN. Bars are means ± SEM (n = 116–121 cells per condition); *P
Figure Legend Snippet: Expression of GIT1-C affects cell morphology and the distribution of endogenous liprin-α1. (A) COS7 cells transfected for one day with either FLAG-GIT1, FLAG-GIT1-C, or FLAG-βGalactosidase were re-plated for 1 h on FN. Immunofluorescence for the transfected proteins (FLAG), paxillin, and phalloidin staining for F-actin. Scale bar, 20 µm. Below, 3-fold enlargements of areas from cells stained for paxillin (arrowheads in the corresponding cells above) are shown. (B) Expression of GIT1-C induces a significant increase of cell spreading on FN. Bars are means ± SEM (n = 116–121 cells per condition); *P

Techniques Used: Expressing, Transfection, Immunofluorescence, Staining

12) Product Images from "The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis"

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Journal: Plant Communications

doi: 10.1016/j.xplc.2019.100011

PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.
Figure Legend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

13) Product Images from "The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis"

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Journal: Plant Communications

doi: 10.1016/j.xplc.2019.100011

PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.
Figure Legend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

14) Product Images from "Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity"

Article Title: Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity

Journal: Nature Communications

doi: 10.1038/s41467-019-09411-7

Microtubule (MT) wall-to-end transition in molecular systems combining CENP-E with various MT-associated proteins (MAPs). a Selected time-lapse images of stabilized MTs moving over beads coated with CENP-E motor and the indicated MAP. All proteins were conjugated to beads via anti-GFP antibodies to achieve similar brightnesses, ensuring that any differences in MT interactions are not due to differences in the density of the protein coatings. Numbers are time (min). Arrows show the direction of MT gliding. Bar, 3 µm. Arrowhead in the last EB1 panel points to a loss of tip attachment due to the MT end-to-wall transition. b Quantifications as in Fig. 2c , but for beads coated with mixtures containing the CENP-E motor and the indicated MAP. Data are means ± SEM for results from N independent trials, which are shown with gray dots. For CENP-E only, CENP-E paired with either Ndc80 Broccoli, Ska1 complex, CENP-E Tail, EB1, or CLASP2 N = 4, 7, 3, 4, 3, and 4, respectively. Total number of observed events is indicated below each column. Asterisk above a bar ( p
Figure Legend Snippet: Microtubule (MT) wall-to-end transition in molecular systems combining CENP-E with various MT-associated proteins (MAPs). a Selected time-lapse images of stabilized MTs moving over beads coated with CENP-E motor and the indicated MAP. All proteins were conjugated to beads via anti-GFP antibodies to achieve similar brightnesses, ensuring that any differences in MT interactions are not due to differences in the density of the protein coatings. Numbers are time (min). Arrows show the direction of MT gliding. Bar, 3 µm. Arrowhead in the last EB1 panel points to a loss of tip attachment due to the MT end-to-wall transition. b Quantifications as in Fig. 2c , but for beads coated with mixtures containing the CENP-E motor and the indicated MAP. Data are means ± SEM for results from N independent trials, which are shown with gray dots. For CENP-E only, CENP-E paired with either Ndc80 Broccoli, Ska1 complex, CENP-E Tail, EB1, or CLASP2 N = 4, 7, 3, 4, 3, and 4, respectively. Total number of observed events is indicated below each column. Asterisk above a bar ( p

Techniques Used:

15) Product Images from "Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration"

Article Title: Paxillin-Kinase-Linker Tyrosine Phosphorylation Regulates Directional Cell Migration

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E09-07-0548

Tyrosine phosphorylation of PKL regulates its interaction with paxillin. (A) MEFs were transfected with GFP-PKL WT or GFP-PKL 3YF. Quiescent cells were stimulated with PDGF (20 ng/ml) at indicated time points, and exogenous PKL was precipitated with GFP
Figure Legend Snippet: Tyrosine phosphorylation of PKL regulates its interaction with paxillin. (A) MEFs were transfected with GFP-PKL WT or GFP-PKL 3YF. Quiescent cells were stimulated with PDGF (20 ng/ml) at indicated time points, and exogenous PKL was precipitated with GFP

Techniques Used: Transfection

PKL phosphorylation and interaction with paxillin regulates Golgi reorientation in migrating cells. (A and B) MEFs expressing GFP-PKL WT, 3YF, ΔPBS2, GFP-paxillin WT, and ΔLD4 were cultured to confluency. Cells were scraped and cultured
Figure Legend Snippet: PKL phosphorylation and interaction with paxillin regulates Golgi reorientation in migrating cells. (A and B) MEFs expressing GFP-PKL WT, 3YF, ΔPBS2, GFP-paxillin WT, and ΔLD4 were cultured to confluency. Cells were scraped and cultured

Techniques Used: Expressing, Cell Culture

PKL tyrosine phosphorylation is required for polarized localization of βPIX to the leading edge. (A and B) NIH 3T3 cells transfected with GFP-PKL WT or 3YF (in green) were fixed and stained with βPIX (in red) and paxillin (in blue) 1 h
Figure Legend Snippet: PKL tyrosine phosphorylation is required for polarized localization of βPIX to the leading edge. (A and B) NIH 3T3 cells transfected with GFP-PKL WT or 3YF (in green) were fixed and stained with βPIX (in red) and paxillin (in blue) 1 h

Techniques Used: Transfection, Staining

16) Product Images from "Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics"

Article Title: Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200608010

In the absence of FAK, PKL and PAK activity are diminished at FAs. (A) Immunoblot analyses reveal reduced levels of PKL without major differences in levels of PAK or PAK-interacting guanine nucleotide exchange factor (βPIX; actin is control). (B) Immunofluorescence microscopy shows that PKL, βPIX, PAK, and phosphorylated (active) PAK (p-PAK) localize at FAs in WT MKs, whereas Fak KO FAs still contain βPIX but show severely reduced staining for PKL, PAK, and p-PAK. Phalloidin (red) marks F-actin, and DAPI (blue) marks chromatin.
Figure Legend Snippet: In the absence of FAK, PKL and PAK activity are diminished at FAs. (A) Immunoblot analyses reveal reduced levels of PKL without major differences in levels of PAK or PAK-interacting guanine nucleotide exchange factor (βPIX; actin is control). (B) Immunofluorescence microscopy shows that PKL, βPIX, PAK, and phosphorylated (active) PAK (p-PAK) localize at FAs in WT MKs, whereas Fak KO FAs still contain βPIX but show severely reduced staining for PKL, PAK, and p-PAK. Phalloidin (red) marks F-actin, and DAPI (blue) marks chromatin.

Techniques Used: Activity Assay, Immunofluorescence, Microscopy, Staining

17) Product Images from "The Cell Adhesion-associated Protein Git2 Regulates Morphogenetic Movements during Zebrafish Embryonic Development"

Article Title: The Cell Adhesion-associated Protein Git2 Regulates Morphogenetic Movements during Zebrafish Embryonic Development

Journal: Developmental biology

doi: 10.1016/j.ydbio.2010.10.027

Identification and characterization of git2 genes in zebrafish ( A ) Phylogenetic analysis of zebrafish git2 family genes. Dendogram of zebrafish git2a on chromosome 5 and git2b on chromosome 10 and related orthologs from other species. ( B ) in situ hybridization of git2a mRNA expression in the zebrafish embryo. git2a expression was ubiquitously detected at the 4-cell, epiboly, tailbud and 14-somite (14SS) and 24hpf stages. ( C ) Western blotting of zebrafish Git2 protein at dome, 50%, 75%, 90% epiboly and 6-somite (6SS) stages, α-Tubulin and paxillin were used as loading controls. ( D ) Immunohistochemistry of Git2 (red) at the 75% epiboly stage. Embryos were co-stained with phalloidin to detect F-actin (green). Images show surface EVL cells and deep cells (30μm below the surface). Scale bar, 50μm. Fluorescent intensity profiles show relative F-actin (green) and Git2 (red) levels in EVL cells at the blastoderm margin (1) and deep cells (2). ( E .
Figure Legend Snippet: Identification and characterization of git2 genes in zebrafish ( A ) Phylogenetic analysis of zebrafish git2 family genes. Dendogram of zebrafish git2a on chromosome 5 and git2b on chromosome 10 and related orthologs from other species. ( B ) in situ hybridization of git2a mRNA expression in the zebrafish embryo. git2a expression was ubiquitously detected at the 4-cell, epiboly, tailbud and 14-somite (14SS) and 24hpf stages. ( C ) Western blotting of zebrafish Git2 protein at dome, 50%, 75%, 90% epiboly and 6-somite (6SS) stages, α-Tubulin and paxillin were used as loading controls. ( D ) Immunohistochemistry of Git2 (red) at the 75% epiboly stage. Embryos were co-stained with phalloidin to detect F-actin (green). Images show surface EVL cells and deep cells (30μm below the surface). Scale bar, 50μm. Fluorescent intensity profiles show relative F-actin (green) and Git2 (red) levels in EVL cells at the blastoderm margin (1) and deep cells (2). ( E .

Techniques Used: In Situ Hybridization, Expressing, Western Blot, Immunohistochemistry, Staining

18) Product Images from "The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis"

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Journal: Plant Communications

doi: 10.1016/j.xplc.2019.100011

LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.
Figure Legend Snippet: LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.

Techniques Used: Chromatin Immunoprecipitation

PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.
Figure Legend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.
Figure Legend Snippet: LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.

Techniques Used: Expressing, Sequencing

19) Product Images from "The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis"

Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

Journal: Plant Communications

doi: 10.1016/j.xplc.2019.100011

LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.
Figure Legend Snippet: LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.

Techniques Used: Chromatin Immunoprecipitation

PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.
Figure Legend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

Techniques Used: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.
Figure Legend Snippet: LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.

Techniques Used: Expressing, Sequencing

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    Becton Dickinson pkl lux interaction
    <t>LUX</t> and <t>PKL</t> Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.
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    LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.

    Journal: Plant Communications

    Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

    doi: 10.1016/j.xplc.2019.100011

    Figure Lengend Snippet: LUX and PKL Regulate H3K27me3 Levels at the DOG1 Locus. (A) ChIP assay. PKL antibody was used to pull down different fragments of DOG1 (shown in Figure 2 C) and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. (B) ChIP assay. H3K27me3 antibody was used to pull down different fragments of DOG1 and the ACT2 control from Col-0, lux-6 , and pkl-1 plants. Seedlings were grown under LD conditions for 5 d and samples were harvested at ZT4. Relative enrichment using the H3K27me3 antibody was normalized to that using the H3 antibody. In all experiments, values denote average ± SD of three biological replicates.

    Article Snippet: To confirm the PKL-LUX interaction, we fused LUX with the B42 activation domain (AD) and full-length PKL or various PKL fragments with the LexA DNA-binding domain (BD) ( A).

    Techniques: Chromatin Immunoprecipitation

    PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

    Journal: Plant Communications

    Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

    doi: 10.1016/j.xplc.2019.100011

    Figure Lengend Snippet: PKL Interacts with LUX. (A) Diagram of the PKL domains and various deletions. Numbers indicate amino acid positions. (B) Yeast two-hybrid assay. Full-length PKL and its deletion variants were fused with the LexA DNA-binding domain (BD-fusion), and LUX, ELF3, and ELF4 were tagged with the B42 activation domain (AD-fusion). Blue colonies denote protein–protein interactions. (C and D) Pull-down assay. D6-His recombinant protein was incubated with GST-LUX (C) or MBP-LUX (D) and immunoprecipitated by anti-GST or anti-MBP antibodies, respectively. (E) LCI assay. Full-length PKL was fused in-frame with the N terminus of LUC and LUX, ELF3, and ELF4 were fused in-frame the C terminus of LUC. Different plasmid compositions were cotransformed into N. benthamiana leaves.

    Article Snippet: To confirm the PKL-LUX interaction, we fused LUX with the B42 activation domain (AD) and full-length PKL or various PKL fragments with the LexA DNA-binding domain (BD) ( A).

    Techniques: Y2H Assay, Binding Assay, Activation Assay, Pull Down Assay, Recombinant, Incubation, Immunoprecipitation, Plasmid Preparation

    LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.

    Journal: Plant Communications

    Article Title: The Evening Complex and the Chromatin-Remodeling Factor PICKLE Coordinately Control Seed Dormancy by Directly Repressing DOG1 in Arabidopsis

    doi: 10.1016/j.xplc.2019.100011

    Figure Lengend Snippet: LUX and ELF3 Affect Circadian Output to Seeds. (A) Relative DOG1 expression in seedlings under free-running conditions. Seedlings were grown under 12 h light/12 h dark for 6 d followed by CL illumination for 24 h. Samples were harvested every 4 h from ZT24. (B) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (6 d after pollination) were harvested every 4 h started from ZT0. (C) Seed germination rate. Col-0, lux , and elf3 plants were grown under LD conditions for 3 weeks and transferred to CL or kept at LD until seed maturation. Germination of freshly harvested seeds in the light was analyzed. (D) Relative DOG1 expression in developing siliques. Plants were grown under LD conditions, and siliques (8 d after pollination) were harvested at ZT8. For (A) , (B) , and (D) , data are the average ± SD of three biological replicates. (E) A working model illustrating the roles of PKL and EC in controlling seed dormancy. LUX binds directly to a specific DNA sequence of DOG1 and recruits PKL to the DOG1 locus through their physical interaction. This interaction increases H3K27me3 levels on DOG1 chromatin, thereby repressing its transcription and leading to reduced seed dormancy. Arrow indicates positive regulation and bar denotes negative regulation.

    Article Snippet: To confirm the PKL-LUX interaction, we fused LUX with the B42 activation domain (AD) and full-length PKL or various PKL fragments with the LexA DNA-binding domain (BD) ( A).

    Techniques: Expressing, Sequencing