Fig. 6.

Unpalmitoylated β4 fails to reach the cell surface. a PC3 cells expressing control vector or DHHC3 knockdown vector were treated with or without Pepstatin A (for 24 h) before labeling with [³H]palmitate for 2 h. Intact cells were then incubated with anti-β4 mAb, washed, and lysed. Cell-surface β4 complexes were then captured using protein G beads, and β4 was detected by [³H] labeling (top panel) and also β4 (middle panel), and α6 (bottom panel) were detected by immunoblotting (lanes 1–4). After removal of cell-surface proteins, the remaining lysate (containing intracellular β4) was immunoprecipitated using anti-β4 mAb, and intracellular proteins were visualized (lanes 4–8). b PC3 cells, with or without Pepstatin A treatment, with control or DHHC3 stable knockdown vectors, were lysed in 1% Triton X-100 buffer. Samples were then blotted for β4 phosphorylated at S1424 and S1356 and the same membranes were blotted for total β4 (2nd and 4th panels) after stripping. Density units in the bar graph represent phospho β4 (S1424)/Total β4 and β4 (S1356)/Total β4. c [³H]palmitate-labeled proteins in PC3 whole cell lysates are visualized