FIGURE 5.

TRIM12c and TRAF6 interact with each other and cooperatively stimulate type I IFN and NF-κB transcription. (A and B) 293T cells (1 × 10⁶) were transfected with wild-type TRIM12c or TRIM12c with the RING domain deletion (TRIM12c ΔR, 2 μg) with or without TRAF6 vector (2 μg) for 30 h, and reporter activity was measured as in Fig. 3. The values represent the average of three independent experiments ± SD. (C) 293T cells (1 × 10⁶) were cotransfected with the expression vectors pUNO-TRAF6 (2 μg), pcDNA3.1 V5-TRIM12c (2 μg), and TRIM12c C15S (2 μg) for 30 h. Whole-cell extracts (WCE) were imunoprecipitated with anti-TRAF 6 Ab and immunoblotted for TRAF6 or V5-TRIM12c. In lower panels, WCE were immunoblotted for TRAF6 or V5-TRIM12. The position of TRAF6 and TRIM12c is indicated in the right. (D) 293T cells (2 × 10⁶) were cotransfected with pcDNA3.1-Flag-IRF3 (4 μg), pcDNA3.1 V5-TRIM12c (4 μg), TRIM12c C15S (4 μg), pUNO-TRAF6 (4 μg), and pUNO-TRAF6 C70S (4 μg) for 36 h, and WCE were immunoblotted with indicated Ab. (E) BM-derived macrophages (2 × 10⁶) expressing Trim12c or control (Ctrl) shRNA were stimulated by poly(I:C) (100 μg/ml) for 8 h. WCE (50 μg) were immunoblotted with indicated Ab.