Fig. 4. Metabolic labelling of RhoA during hypoxia is localized to membranes. Caki-1 cells were pre-incubated in cystein and methionine free medium for 1 hour, then incubated with 50 µCi/ml of [³⁵S] Met/Cys. Subsequently, cells under normoxia or hypoxia at 1% O₂ for 4 hours were fractionated in soluble fractions (S) and crude membranes (M). RhoA was immunoprecipitated, analyzed by SDS-PAGE, and labelled RhoA detected by autoradiography (A). As a control, Caki-1 cells were exposed to normoxia or hypoxia for 4 hours, fractionated in soluble and membrane fractions, then analyzed by western blot and immunodetected with RhoA antibody (A). Densitometric analysis of immunoprecipitated RhoA from cells under normoxia () and hypoxia () conditions is represented (B). Means ± s.e.m. are shown for two different experiments relative to normoxia. Significant differences (P<0.05) from normoxia are indicated by asterisks (*).