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<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 "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

4) 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

5) 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

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

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

7) 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

8) 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

9) 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

10) 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

11) 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

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Flow Cytometry:

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Cytometry:

Article Title: Anti-GITR Antibody Treatment Increases TCR Repertoire Diversity of Regulatory but not Effector T Cells Engaged in the Immune Response Against B16 Melanoma
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Blocking Assay:

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Staining:

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Article Title: Early IL-6 signalling promotes IL-27 dependent maturation of regulatory T cells in the lungs and resolution of viral immunopathology
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IA:

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FACS:

Article Title: Satb1 regulates the effector program of encephalitogenic tissue Th17 cells in chronic inflammation
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Article Title: Satb1 regulates the effector program of encephalitogenic tissue Th17 cells in chronic inflammation
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