Fig. 4. Increased stability of R664X AE1 in K562 cells in the presence of proteasome inhibitors. (A) K562bebWT and K562bebRX cells were incubated for 8 hours in the absence (None) or presence of the reagents indicated, and then wild-type and R664X protein levels were analyzed by immunoblotting with the tmb3-29 antibody. (B) K562bebWT and K562bebRX cells were pulse-labeled and chased for the indicated periods followed by immunoprecipitation of wild-type (WT) or R664X (R664X) AE1 in the presence (Lactacystin, ) or absence (None, ) of 10 µM lactacystin, as described in the legend for Fig. 1. (C) K562bebWT (WT) and K562bebRX (RX) cells were incubated for 8 hours in the absence (-) or presence (+) of 10 µM lactacystin and lysed in IP-buffer, followed by immunoprecipitation with cdb3-64. AE1 proteins and ubiquitylated proteins in detergent-soluble whole cell lysates (Whole, upper panels) or immunoprecipitates (IP, lower panels) were detected with the anti-AE1 (left) and anti-ubiquitin (right) antibodies, respectively. The detergent-insoluble fraction contained various ubiquitylated polypeptides but not proteins recognized with the AE1 antibody (data not shown). Blots were incubated with tmb3-29 (Whole) or anti-38K (IP) antibodies. Asterisks indicate non-specific signals observed in lysates from mock-treated cells (not shown). Migrating positions of the protein standards are shown in kDa.