FIGURE 4.

Point mutant of FAK is resistant to calpain proteolysis. A, HEK 293 cells were transfected with wild-type GFP-FAK (WT), GFP-FAK-V744G, or GFP-FAKΔ724–750. Cells were treated with vehicle (−), 50 μg/ml calpain inhibitor N-acetyl-Leu-Leu-Met (ALLM), or 1 μm ionomycin and 10 mm CaCl₂. Cell lysates were analyzed by immunoblotting and probed for FAK. The immunoblot shown is from one of three independent experiments. C-term, C-terminal. B, cell lysates from HEK 293 cells transiently transfected with GFP-FAK or GFP-FAK-V744G were analyzed by immunoblotting and probed with anti-GFP and anti-HA antibodies. Immunoblots shown are from one of five independent experiments. Quantification of relative cleavage is defined as the ratio of cleaved GFP-FAK to total GFP-FAK (full-length + cleaved) relative to GFP-FAK. Relative cleavage is shown as the mean ± S.E. *, p < 0.05 (by t test) compared with wild-type GFP-FAK. C, HEK 293 cells were transiently transfected with GFP-FAK or GFP-FAK-V744G. Proteins were immunoprecipitated (IP) with anti-HA antibody, followed by immunoblotting with anti-HA and anti-phospho-Tyr-397 (pY397) FAK antibodies. Immunoblots shown are from one of three independent experiments.