FIG. 9.

The RING domain regulates the localization of Siah-1 in cells. COS-1 cells were transiently transfected with expression plasmids encoding Flag-Siah-1 (A), Flag-Siah-1-dR (B), or an empty pcDNA3 vector as a control (C). Forty-eight hours after transfection, cells were incubated with Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and 50 μM MG132 for 4 h at 37°C and then fixed. Cellular localization of the expressed proteins was detected by indirect immunofluorescence staining of the cells with anti-Flag M2 monoclonal antibody and a FITC-conjugated goat anti-mouse immunoglobulin secondary antibody. Cells were also stained with DAPI to visualize the nuclei.