Fig. 3.

The α1(A322D) subunit was degraded through the ubiquitin–proteasome system. (A) Mock-transfected cells (m) or those transfected with the α1(A322D)-FLAG subunit were incubated for 30 min in the absence (−, ■) or presence (+, □) of 10 μM lactacystin (LAC). The cells were then [³⁵S]methionine pulse-labeled for 10 min and then chased for the indicated time periods (n = 4). Statistical significance was indicated by an asterisk. (B) Mock-transfected cells or cells transfected with α1-FLAG (a) or α1(A322D)-FLAG (d) subunits were incubated in the absence or presence of 10 μM lactasystin (n ≥ 5) and analyzed by immunoblot that was probed with an antibody directed against polyubiquitin (B1) or FLAG (B2). The relative amounts of polyubiquitinated subunits were plotted (B3).