Figure 6.

Rab4 is in a complex with Pin1 in cytosol during mitosis. Equal numbers of interphase and mitotic rab4/HisPin1 CHO double transfectants and rab4 CHO cells (A, B), or rab4S196Q and rab4S196Q/HisPin1 CHO transfectants (B) were homogenized and fractionated by high speed centrifugation. Cytosol was retrieved and incubated with 50 μl Ni-NTA beads. Beads were washed three times with ice-cold RIPA buffer and boiled in Laemmli sample buffer. Eluted proteins were separated by SDS-PAGE and analyzed by Western blot using the Xpress antibody for HisPin1 and a rabbit rab4 antibody. Detection was done with HRP-labeled secondary antibodies and enhanced chemiluminescence and quantitated using the NIH image software package.