FIGURE 7.

Palmitoylation of G5. (A) Recombinant G5 and G8 were transiently expressed in CRL-1601 hepatocytes. A total of 48 h after transfection of the expression plasmids, the cells were metabolically labeled with [³H]-palmitic acid. The cells were solubilized in 1 mL of lysis buffer, and G5 and G8 were immunoprecipitated using anti-G5 and anti-G8 specific antibodies. Immunoprecipitated samples were separated by 8% SDS–PAGE followed by immunoblotting or X-ray film exposure (7 days). (B) Labeled cells were washed once with 20 mL of ice-cold PBS, broken by passing through a 25-gauge needle 20 times, and then treated with 1 M hydroxylamine (pH 7.0) or 1 M Tris-HCl (pH 7.0) for 2 h at 37 °C, followed by IP, SDS–PAGE, and Western blotting or X-ray film exposure. (C) Site-directed mutagenesis was used to substitute selected cysteines predicted to be located in the cytoplasmic regions of G5 with alanine. The effect of these substitutions on palmitoylation was assayed as described above.