FIG. 4.

Regulation of Siah-1 protein expression by the proteasome pathway. (A) Increased expression of wild-type Siah-1 expression, but not the RING-deleted form, following treatment of cells with the proteasome inhibitor MG132. cDNAs encoding Flag-tagged wild-type Siah-1 and the RING-deleted form of Siah-1 (SIAH1-dR) were transfected into COS-1 cells, as indicated. Forty-eight hours after transfection, the cells were treated for 6 h with various concentrations of MG132, as indicated. Cell lysates were prepared, and ECL-Western blot analysis with the anti-Flag M2 antibody was performed. The blot was then stripped and reprobed with anti-actin polyclonal antibody C11 to verify the loading. The migration positions (in kilodaltons) of selected markers are shown to the left of the blots. (B) Expression of Siah-1 proteins with missense mutations is also increased following treatment of cells with MG132. Studies essentially identical to those in panel A were carried out. A no DNA “mock” negative-control transfection is shown in lane 9. The migration positions (in kilodaltons) of selected protein markers are shown to the left of the blots.