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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Images

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 "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

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) 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

Related Articles

Western Blot:

Article Title: Control of mitochondrial function and cell growth by the atypical cadherin Fat1
Article Snippet: .. We used antibodies against Fat1 (ref. ) (1:10,000 for western blot (WB), 1:2,000 for immunofluorescence (IF), 1:2,000 for immunohistochemistry (IHC)), BiP (1:5,000 for WB, 610979, BD Biosciences), Flag (1:15,000 for WB, F3165, Sigma), cyclin D1 (1:1,000 for WB, 1:200 for IHC, 2978S, Cell Signaling), total OXPHOS cocktail (1:1,000 for WB, ab110413, Abcam), LC3 I/II (1:1,000 for WB, 2775S, Cell Signaling), complex I NDUFA9 (1:1,000 for WB, ab14713, Abcam), complex II 70 kDa Fp subunit (1:10,000 for WB, 459200, Invitrogen), complex III subunit core 1 (1:2,000 for WB, 459140, Invitrogen), complex IV MTCO1 (1:2,000 for WB, ab14705, Abcam), complex V ATP synthase subunit α (1:1,000 for WB, 459240, Life Technologies), ACTA2 (1:200 for IHC, M0851 Clone 184, DAKO), phospho-histone H3 (1:150 for IHC, 9701, Cell Signaling), transgelin (1:250 for IF, ab14106, Abcam), calponin 1 (1:200 for IF, ab46794, Abcam), pecam1 (1:200 for IF, ab28364, Abcam), anti-mouse IgG HRP (1:10,000, 115-036-072, Jackson ImmunoResearch Labs), Alexa Fluor 488 conjugated anti-rabbit (1:250, 111-545-047, Jackson ImmunoResearch), Alexa Fluor 546 conjugated anti-mouse (1:250, A-11003, Molecular Probes). .. The following antibodies were from Santa Cruz: complex I NDUFS3 (1:600 for WB, sc374282), c-Myc (1:20,000 for WB, sc789), β-catenin (1:800 for WB, sc7963), PGC1-α (1:150 for WB, sc13067), PGC1-β (1:100 for WB, sc373771), Tfam (1:150 for WB, sc23588), Mfn1 (1:600 for WB, sc50330), Mfn2 (1:600 for WB, sc50331), Acta2 (1:200 for IF, sc32351), nitrotyrosine (1:250 for IF, sc32757), GAPDH (1:8,000 for WB, sc25778), anti-rabbit IgG HRP (1:10,000, sc2030), and anti-goat IgG HRP (1:10,000, sc2056).

Incubation:

Article Title: Amelioration of salt-induced vascular dysfunction in mesenteric arteries of Dahl salt-sensitive rats by missense mutation of extracellular superoxide dismutase
Article Snippet: .. The membrane was incubated at 4°C overnight with primary antibodies in 2% nonfat dry milk in Tris-buffered saline-Tween (TBST) buffer: 1 ) ecSOD (Santa Cruz) at 1:3,000; 2 ) Cu/Zn SOD (Enzo Life Sciences) at 1:6,000; 3 ) eNOS (BD Biosciences) at 1:3,000; 4 ) p-eNOS (BD Biosciences) at 1:1,000; and 5 ) β-actin (Sigma) at 1:35,000. .. Secondary horseradish peroxidase-conjugated antibodies in 2% nonfat dry milk in TBST buffer were applied at room temperature for 2 h. These included goat anti-rabbit (Sigma) at 1:6,000 for Cu/Zn SOD; goat anti-mouse (Sigma) at 1:35,000 for β-actin; and goat anti-mouse (Sigma) at 1:2,000 for ecSOD, Cu/Zn SOD, eNOS, and p-eNOS.

Article Title: Wogonin suppresses the LPS-enhanced invasiveness of MDA-MB-231 breast cancer cells by inhibiting the 5-LO/BLT2 cascade
Article Snippet: .. The membrane was exposed to TBS containing 0.05% Tween-20 and 5% dried nonfat milk for 1 h and then incubated with primary antibodies against 5-LO (cat. no. 610695; 1:1,000; BD Transduction Laboratories Inc.; BD Biosciences), MMP-9 (cat. no. CST 3852), p-ERK (cat. no. 9101S), total ERK (cat. no. 9102) and β-actin (cat. no. 4970S) (1:1,000; all from Cell Signaling Technology, Inc.). .. Secondary antibodies were anti-rabbit (cat. no. 7074S; 1:2,000) or anti-mouse antibodies (cat. no. 7076S, 1:2,000) both from Cell Signaling Technology, Inc., and then, the proteins were visualized using enhanced chemiluminescence reagent (Amersham; GE Healthcare, Chicago, IL, USA), according to the manufacturer's recommendations.

Immunohistochemistry:

Article Title: Lkb1 regulates granule cell migration and cortical folding of the cerebellar cortex
Article Snippet: .. The following antibodies were used for immunohistochemistry: p27Kip1 (BD Biosciences, 1:300), Tag1 (Hybridoma Bank, 1:10), γ-tubulin (Sigma, 1:300), BrdU (Hybridoma Bank, 1:100), Ki67 (Thermo Scientific, 1:200), phosphohistone H3 (Upstate, 1:300), p-S6 (Cell Signaling, 1:200), Aurora B (BD Biosciences, 1:300), ARL13B (a kind gift from Jonathan Eggenschwiler, 1:5000), Keratin (Sigma, 1:250), N-Cadherin (Hybridoma Bank, 1:200), Lkb1 (Santa Cruz, 1:200). .. EdU was purchased from Molecular Probes.

Article Title: Control of mitochondrial function and cell growth by the atypical cadherin Fat1
Article Snippet: .. We used antibodies against Fat1 (ref. ) (1:10,000 for western blot (WB), 1:2,000 for immunofluorescence (IF), 1:2,000 for immunohistochemistry (IHC)), BiP (1:5,000 for WB, 610979, BD Biosciences), Flag (1:15,000 for WB, F3165, Sigma), cyclin D1 (1:1,000 for WB, 1:200 for IHC, 2978S, Cell Signaling), total OXPHOS cocktail (1:1,000 for WB, ab110413, Abcam), LC3 I/II (1:1,000 for WB, 2775S, Cell Signaling), complex I NDUFA9 (1:1,000 for WB, ab14713, Abcam), complex II 70 kDa Fp subunit (1:10,000 for WB, 459200, Invitrogen), complex III subunit core 1 (1:2,000 for WB, 459140, Invitrogen), complex IV MTCO1 (1:2,000 for WB, ab14705, Abcam), complex V ATP synthase subunit α (1:1,000 for WB, 459240, Life Technologies), ACTA2 (1:200 for IHC, M0851 Clone 184, DAKO), phospho-histone H3 (1:150 for IHC, 9701, Cell Signaling), transgelin (1:250 for IF, ab14106, Abcam), calponin 1 (1:200 for IF, ab46794, Abcam), pecam1 (1:200 for IF, ab28364, Abcam), anti-mouse IgG HRP (1:10,000, 115-036-072, Jackson ImmunoResearch Labs), Alexa Fluor 488 conjugated anti-rabbit (1:250, 111-545-047, Jackson ImmunoResearch), Alexa Fluor 546 conjugated anti-mouse (1:250, A-11003, Molecular Probes). .. The following antibodies were from Santa Cruz: complex I NDUFS3 (1:600 for WB, sc374282), c-Myc (1:20,000 for WB, sc789), β-catenin (1:800 for WB, sc7963), PGC1-α (1:150 for WB, sc13067), PGC1-β (1:100 for WB, sc373771), Tfam (1:150 for WB, sc23588), Mfn1 (1:600 for WB, sc50330), Mfn2 (1:600 for WB, sc50331), Acta2 (1:200 for IF, sc32351), nitrotyrosine (1:250 for IF, sc32757), GAPDH (1:8,000 for WB, sc25778), anti-rabbit IgG HRP (1:10,000, sc2030), and anti-goat IgG HRP (1:10,000, sc2056).

Immunofluorescence:

Article Title: USP9X Limits Mitotic Checkpoint Complex Turnover to Strengthen the Spindle Assembly Checkpoint and Guard against Chromosomal Instability
Article Snippet: .. The following primary antibodies were used for immunofluorescence: aurora B (1/1,000, AIM-1, 611082; BD Biosciences); phospho-Thr232-aurora B (1/2,000, 600-401-677; Rockland); BubR1 (1/1,000, A300-386A) and Mad2 (1/1,000, A300-301A; Bethyl Laboratories); Mad1 (1/1,000, MABE867; Millipore); Cenp-C (1/1,000, PD-030; Caltag Medsystems); ACA/CREST (1/1,000, FZ90C-CS1058; Europa Bioproducts); and KNL1-pMELT13/17 (1/2,000, ). .. Secondary antibodies were Alexa Fluor 647 goat-anti-guinea pig, Alexa Fluor 488 donkey anti-rabbit, and Alexa Fluor 568 goat anti-mouse (Invitrogen) for immunofluorescence and anti-mouse/rabbit-HRP (Bio-Rad) for western blotting (all at 1/1,000).

Article Title: Lipidome of midbody released from neural stem and progenitor cells during mammalian cortical neurogenesis
Article Snippet: .. The following primary antibodies were used: rabbit anti-Prominin-1 [αD, MPI-CBG; 1:20,000 for immunoblotting (IB)]; goat antibody against apolipoprotein A1 (Biodesign; 1:1000); mouse monoclonal antibodies against CRIK [BD bioscience; 1:200 for immunofluorescence (IF) and 1:4000 for IB], α-tubulin (Sigma; 1:300 for IF and 1:60,000 for IB) and Aurora B (BD bioscience; 1:300) and rat anti-Prominin-1 (13A4, MPI-CBG; 1:300 for IF). .. Secondary antibodies were: horseradish peroxidase-coupled antibodies (Jackson laboratory), Alexa Fluor 488, 555, and 647 labeled IgG antibodies (Invitrogen).

Article Title: Control of mitochondrial function and cell growth by the atypical cadherin Fat1
Article Snippet: .. We used antibodies against Fat1 (ref. ) (1:10,000 for western blot (WB), 1:2,000 for immunofluorescence (IF), 1:2,000 for immunohistochemistry (IHC)), BiP (1:5,000 for WB, 610979, BD Biosciences), Flag (1:15,000 for WB, F3165, Sigma), cyclin D1 (1:1,000 for WB, 1:200 for IHC, 2978S, Cell Signaling), total OXPHOS cocktail (1:1,000 for WB, ab110413, Abcam), LC3 I/II (1:1,000 for WB, 2775S, Cell Signaling), complex I NDUFA9 (1:1,000 for WB, ab14713, Abcam), complex II 70 kDa Fp subunit (1:10,000 for WB, 459200, Invitrogen), complex III subunit core 1 (1:2,000 for WB, 459140, Invitrogen), complex IV MTCO1 (1:2,000 for WB, ab14705, Abcam), complex V ATP synthase subunit α (1:1,000 for WB, 459240, Life Technologies), ACTA2 (1:200 for IHC, M0851 Clone 184, DAKO), phospho-histone H3 (1:150 for IHC, 9701, Cell Signaling), transgelin (1:250 for IF, ab14106, Abcam), calponin 1 (1:200 for IF, ab46794, Abcam), pecam1 (1:200 for IF, ab28364, Abcam), anti-mouse IgG HRP (1:10,000, 115-036-072, Jackson ImmunoResearch Labs), Alexa Fluor 488 conjugated anti-rabbit (1:250, 111-545-047, Jackson ImmunoResearch), Alexa Fluor 546 conjugated anti-mouse (1:250, A-11003, Molecular Probes). .. The following antibodies were from Santa Cruz: complex I NDUFS3 (1:600 for WB, sc374282), c-Myc (1:20,000 for WB, sc789), β-catenin (1:800 for WB, sc7963), PGC1-α (1:150 for WB, sc13067), PGC1-β (1:100 for WB, sc373771), Tfam (1:150 for WB, sc23588), Mfn1 (1:600 for WB, sc50330), Mfn2 (1:600 for WB, sc50331), Acta2 (1:200 for IF, sc32351), nitrotyrosine (1:250 for IF, sc32757), GAPDH (1:8,000 for WB, sc25778), anti-rabbit IgG HRP (1:10,000, sc2030), and anti-goat IgG HRP (1:10,000, sc2056).

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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