Figure 4 - full size



	Figure 4 - full size

Figure 4.
Figure 4. R197A-R198A double mutation reduces the efficiency of PINCH localization to focal adhesions. ILK binding as analyzed by western blotting with (a) HRP-conjugated anti-GFP and (b) mouse monoclonal anti-ILK 65.1. Lysates (8 g per lane) of C2C12 cells expressing GFP-FLAG -tagged PINCH, the GFP-FLAG -tagged PINCH mutant with the R197A-R198A mutation (PINCHm) or GFP alone (control) were mixed with rabbit polyclonal antibodies to GFP (Clontech) and then immunoprecipitated as indicated. (c -f) Subcellular localization. C2C12 cells expressing GFP-FLAG-PINCHm (c and d) or GFP-FLAG-PINCH (e and f) were plated on fibronectin-coated coverslips and stained with mouse monoclonal antibody to paxillin (a marker of focal adhesions; clone 349, Transduction Laboratories) and rhodamine-conjugated anti-mouse IgG. GFP-FLAG-PINCHm (c), GFP-FLAG-PINCH (e) and paxillin (d and f) were visualized under a fluorescence microscope equipped with GFP (c and e) and rhodamine (d and f) filters. Bar, 10 m. Although the GFP-FLAG-PINCH mutant and GFP-FLAG-PINCH proteins bind to ILK equally well, the GFP-FLAG-PINCH mutant localized to focal adhesions much less efficiently than GFP-FLAG-PINCH.