Figure 7.

Phosphorylated rab4 is associated with Pin1 during mitosis. Equal numbers of mitotic CHO cells expressing rab4, NHrab4S22N, NHrab4Q67L, rab4/HisPin1 or control CHO cells were labeled for 45 min with 500 μCi/ml ³²P ortho phosphate and lysed in 1% octylglucoside, 50 mM HEPES pH 7.6, 200 mM NaCl, 10 mM NaF, 25 mM Na-β-glycerophosphate, 1 mM sodium vanadate, 1 mM PMSF, 10 μg/ml aprotinin, 5 μg/ml leupeptin, 10 μg/ml pepstatin A. Detergent lysates were incubated for 1 h with a Pin1 antibody (+) or matched preimmune serum (-) adsorbed to Protein A Sepharose CL-4B and washed 3 times with octylglucoside wash buffer. Washed immunoprecipitates were boiled 5 min with 100 μl 0.5% SDS in PBS and pelleted. Supernatants were used to immunoprecipitate rab4 as described in the methods section, resolved on 12.5% SDS PAA mini gels and analyzed by phosphorimaging (B).