Fig. 5. Spectrin is cleaved by calpain upstream of cluster formation. BAE cells were serum-starved for 16 hours and either solubilized in SDS-containing buffer or allowed to spread on fibronectin-coated dishes for 15, 20 or 30 minutes. (A) Cells were solubilized in SDS-containing buffer and analyzed on western blots with polyclonal antibodies against bovine - and ß-spectrin. (B) BAE cells were allowed to spread on fibronectin-coated coverslips for 30 minutes or 8 hours, fixed and permeabilized. In the top panel, samples were incubated with polyclonal antibodies that detect only a 150 kD calpain-cleaved fragment of -spectrin and monoclonal antibody against calpain (as a marker for the early integrin-containing clusters). In the bottom panel, samples were incubated with antibodies against the calpain-generated fragment of spectrin and with antibodies against vinculin, a marker of focal complexes and focal adhesions. (C) Cells were fixed, permeabilized, and incubated with polyclonal antibodies that detect only the 150 kD calpain-cleaved fragment of spectrin. Arrows indicate spectrin-fragment-containing clusters. (D) Cells were plated for 20 or 30 minutes, fixed, permeabilized and stained with polyclonal antibodies specific for the 150 kD fragment of -spectrin. Confocal analysis was performed on horizontal (x-y plane) optical sections (0.3 µm intervals); for each time point, four sections going from the upper to the lower surface (left to right) of a single cell are shown. Arrows indicate spectrin-fragment-containing clusters. Bars, 20 µm (A-C); 5 µm (D).