hGAAP stimulates calpain activity. (A) U2-OS cells were transfected with the CFP/YFP calpain FRET biosensor, plated on fibronectin, and imaged live over 20 min in both FRET and IRM channels. Typical examples show stills from videos. Right images show enlargements of boxed areas in left images. Bars, 5 µm. (B) Summary results from the images collected at t = 0 (means ± SEM, for >10 cells) show the percentages of low-FRET pixels (ratio < 1.5, i.e., high calpain activity) within 5 µm of the plasma membrane. (C) Typical IB showing total FAK, calpain 2–dependent cleaved FAK (asterisk, FAK(clv)), and the loading control (α-GAPDH) for U2-OS cells transfected as shown, reseeded on fibronectin for 30 min, and assayed with or without treatment with ALLM. (D) Summary results (means ± SEM, n = 3). (E) IB, typical of three independent experiments, showing total endogenous calpain 2. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test, relative to neo or hGAAP cells).