FIGURE 10.

Localization of cysteine mutant Cosmc. A–D, immunofluorescent staining of mutant Cosmc (C19S). Cells were stained with anti-HPC4 (red) antibody and anti-giantin (green) antibodies. Merge, yellow. DAPI, blue. E, sucrose gradient subcellular fractionation. The PNS was applied to a sucrose gradient and 18 fractions (top to bottom) were obtained after ultracentrifugation. Proteins from each fraction were analyzed on Western blot with anti-HPC4 and anti-KDEL antibodies and (F) measured for T-synthase activity. G, COS-7 cells transiently expressing wild-type Cosmc (FL) or mutant Cosmc (C19S and C19A) were extracted by the given protocol and included 250 mm iodoacetamide in the extraction buffer to limit artifactual disulfide-bonded dimerization. After treatment, the cell extracts were analyzed on SDS-PAGE with or without β-ME by Western blot with anti-HPC4 antibody.