Fig. 2. Dominant-negative effect of R664X AE1 on cell surface expression of wild-type AE1. (A) Wild-type () proteins in K562bebWT cells and wild-type () and R664X () proteins in K562bebWT/RX cells were pulse-labeled and chased for the indicated periods as in Fig. 1. The left figure shows immunoprecipitates from each sample after labeling without chase. The asterisk indicates a non-specific band. Data are the mean of duplicate samples at each time point. (B) K562bebWT, K562bebRX, K562bebWT/RX and K562C cells were transfected with bovine GPC and stained for AE1 and GPC with cdb3-64 and the anti-GPC antibody, respectively. Bars, 5 µm. (C) AE1 proteins in K562bebWT (WT), K562bebRX (RX), and K562bebWT/RX (WT/RX) cells were immunoprecipitated with monoclonal antibodies cdb3-64 or tmb3-26 (upper panels). Lane WT + RX contains immunoprecipitates from K562bebWT and K562bebRX cells mixed prior to solubilization for immunoprecipitation. The asterisk indicates what probably is an 70-kDa degradation product of wild-type AE1. Likewise, AE1 proteins synthesized in a cell-free translation system in the presence of canine pancreatic microsomes (Synthesized) were immunoprecipitated with tmb3-26 (lower right panel). Reactions contained wild type alone (WT), R664X alone (R664X), or both wild type and R664X AE1 (WT/RX). WT and RX reactions were combined prior to solubilization (WT + RX).