Journal: Journal of Biological Chemistry

Article Title: Integrin α2-mediated ERK and Calpain Activation Play a Critical Role in Cell Adhesion and Motility via Focal Adhesion Kinase Signaling

doi: 10.1074/jbc.m600787200

Figure Lengend Snippet: FIGURE 2. Adhesion of GEO cells to CN IV induces tyrosine phosphorylation of proteins (A), FAK cleavage (B), FAK dephosphorylation by P1E6 (C), and ERK activation (D). GEO cells were trypsinized, incubated with soy bean trypsin inhibitor for 1 h at 37 °C, and either kept in suspension or replated to dishes coated with CN IV (5 g/ml) in SM medium. Cells were allowed to attach to CN IV for 20 or 60 min at 37 °C. Cellular lysates from cells in suspension (S) and cells attached (A) were analyzed for phosphotyrosine proteins by immunoblotting with antiphosphotyrosine antibody 4G10 (A). The immunoblots were analyzed for activation of FAK (B; upper left) or for FAK protein by using rabbit anti-human N-terminal FAK antibody (B; lower left). The right panel shows that both 125-kDa FAK and its cleaved 90-kDa form are immunoprecipitated and immunoblotted by N-terminal FAK antibody. GEO cells in SM medium were incubated either with mouse IgG1 or with integrin 2-blocking mAb (clone P1E6) at dilution 1:50 for 30 min at 37 °C. Subsequently, cells were transferred to dishes precoated with CN IV and incubated for 1 h at 37 °C. The cell lysates were analyzed by immunoblotting for pFAK and FAK using specific antibodies (C). Cellular lysates from cells in suspension (S) and cells attached (A) were also analyzed for ERK (Tyr204) by immunoblotting with specific antibody (D). Molecular weight standards are indicated in A. The positions of pFAK (125 kDa) and its major cleaved fragment (90 kDa), ERK1 (44 kDa), and ERK2 (42 kDa) are indicated by the arrowheads. Total FAK and ERK panels are shown for equal loading.

Article Snippet: The polyclonal anti-FAK phosphospecific antibodies were provided by BIOSOURCE (Camarillo, CA), whereas the rabbit anti-human C-terminal FAK (SC-558), anti-humanN-terminal FAK (SC-557), ERK, phosphorylated ERK1/2, and cyclin E antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Incubation, Suspension, Western Blot, Immunoprecipitation, Blocking Assay, Molecular Weight

Journal: Journal of Biological Chemistry

Article Title: Integrin α2-mediated ERK and Calpain Activation Play a Critical Role in Cell Adhesion and Motility via Focal Adhesion Kinase Signaling

doi: 10.1074/jbc.m600787200

Figure Lengend Snippet: FIGURE 8. Effect of U0126, cyto D, and dominant negative mutants on ERK activation. GEO cells were treated either with Me2SO or with MEK inhibitor, U0126, for 24 h in SM medium. After trypsinization, cells were seeded on CN IV-coated microarray wells. Cells were allowed to attach for 3 h, and then micromotion was recorded by the ECIS technique. Results show the inhibitory effect of U0126 on micromotion (right) as compared with control (left) (A). B shows the inhibitory effect of U0126 on ERK phosphorylation as determined by Western blot analysis using either phosphospecific antibody (Y204; top) or ERK antibody (bottom). In C, GEO cells were treated either with Me2SO (control) or with cyto D for 24 h. Aftertrypsinization,cellsweretransferredtoCNIV-precoateddishesandincubatedat37 °CinthepresenceofMe2SOorcytoDfor1h.LysateswerecollectedandanalyzedbyWestern blot using an antibody specific to phospho-ERK or total ERK, as indicated. In D–F, GEO cells were transfected either with empty vector or with the dominant negative FRNK, Y861F, or -calpain mutant for 48 h, respectively. The cell lysates were analyzed by using specific antibodies for phospho-ERK (top) or ERK (bottom).

Article Snippet: The polyclonal anti-FAK phosphospecific antibodies were provided by BIOSOURCE (Camarillo, CA), whereas the rabbit anti-human C-terminal FAK (SC-558), anti-humanN-terminal FAK (SC-557), ERK, phosphorylated ERK1/2, and cyclin E antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Dominant Negative Mutation, Activation Assay, Microarray, Control, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis