Figure 8

Identification of α1- and α7-subunits by western blot analysis. (A) The 20S proteasomes were subjected to SDS-PAGE in a 12% polyacrylamide gel and electrophoretically transferred to a PVDF membrane. The α1- and α7-subunits were detected using their respective antibodies as described in the methods. (B) Cells from wild-type and the rpt6-1 mutant (wt, rpt6-1) grown at 25°C (OD_{600 nm} = 1.0) were harvested and the cell extracts were applied to a Q-sepharose column. Active Q-sepharose fractions were concentrated and treated with (AP+) or without (AP-) alkaline phosphatase as described in the methods. The samples were then subjected to SDS-PAGE in a 12% polyacrylamide gel. The α7-subunit was detected by immunostaining with anti-α7 antibody [26, 27].