FIG.3.

m-Calpain binding to membranes is eliminated by PLC. (A) Quiesced WT NR6 cells were exposed to 1 nM EGF overnight on coverslips, and substratum-associated membranes were prepared. The membrane footprint further treated with or without (dil for diluent) bacterial PLC (100 μg/ml for 10 min at 37°C) was dually stained by m-calpain (green) and PIP₂ (red) antibodies. (B) The footprints were stained simply for F-actin (by antibody, green) to demonstrate that the PLC treatment did not remove the actin cytoskeleton (see also Fig. 2 for total membrane protein). Shown are representative cells from at least three independent experiments for each treatment. (C and D) The specificity of m-calpain binding to PIP₂ was detected by assay of protein-liposome binding. The upper two rows of panels C and D show the immunoblotting of native m-calpain extracted from WT NR6 cells, without and with treatment with EGF, and the bottom row of panel C represents immunoblotting of the D3-eGFP construct. The graphic representation of densitometry scanning in panel C is the average ± the standard deviation of two experiments. Shown in each immunoblot is one of two similar experiments.