FIG. 2.

TNF-R2 recruits ASB3 in cells. (A) c-Myc-ASB3 was transiently transfected into 293 cells, followed by treatment with or without TNF-α for 30 min. Whole extracts prepared from these cells were immunoprecipitated with anti-TNF-R2 followed by Western blot analysis with anti-c-Myc or anti-TNF-R2. (B) 293 cells were transfected with TNF-R2 alone (1 μg pcDNA3 empty vector and 1 μg TNF-R2) or with TNF-R2 and c-Myc-ASB3 (1 μg TNF-R2 and 1 μg c-Myc-ASB3). Forty-eight hours after transfection, cell extracts were prepared for the same immunoprecipitation/Western blot analysis as that described for panel A. As an immunoprecipitation control, immunoglobulin G (normal mouse IgG) was incubated with the extracts prepared from 293 cells expressing TNF-R2 and c-Myc-ASB3. (C) 293 cells were transfected with TNF-R1 alone or with TNF-R1 and c-Myc-ASB3 under similar conditions to those used for panel B. Anti-TNF-R1 precipitates were analyzed for ASB3 binding under similar conditions to those used for panel B. (D) 293 cells were transfected with TNF-R2 (0.1 μg), c-Myc-ASB3 (1 μg), and either empty vector or mTNF-α (0.5 μg). After 48 h, the cells were either left untreated or treated with 10 μM MG132, followed by protein extraction and immunoprecipitation as described for panel A.