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Becton Dickinson anti git pkl
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 <t>GIT-C2.</t> 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 <t>PKL,</t> 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.
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1) 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

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Article Title: Biochemical and Functional Characterization of the Interaction between Liprin-?1 and GIT1: Implications for the Regulation of Cell Motility
Article Snippet: Antibodies The antibodies used in this study were as follows: monoclonal antibodies (mAb) anti-FLAG M5 and M2, anti-talin, and anti-tubulin (Sigma-Aldrich, Saint Louis, MO); anti-HA 12CA5, anti-Myc 9E10 (Primm Biotech, Milano, Italy); anti-paxillin, anti-GIT/PKL, anti-LAR recognizing the 150 kDa form, and mAb 9EG7 recognizing activated human β1 integrins (BD Transduction Laboratories, San Jose, CA).

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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