Figure 3.

Rab4 is phosphorylated on membranes. Mitotic rab4 CHO cells were labeled for the indicated periods of time with 175 μCi/ml ³²P ortho phosphate. At the 45-min time point, labeling medium was removed and cells were incubated with chase medium lacking ³²P ortho phosphate for 0, 10, or 20 min. Cells were fractionated, and rab4 was immunoprecipitated from membrane and cytosol fractions as described in MATERIALS AND METHODS (A). To investigate whether phosphorylated rab4 was tightly associated with membranes, a membrane pellet (m) was resuspended in homogenization buffer containing 1 M NaCl. After 30 min, salt-extracted membranes were reisolated by high-speed centrifugation. Rab4 was immunoprecipitated from cytosol (c), supernatant (w), and washed membranes (wm). Immunoprecipitates were resolved by SDS-PAGE and quantitated by phosphorimaging (B).