Fig. 4. OTUD7b inhibits TRAF2 degradation, increased activation of NF-κB and MAP kinase signaling and expression of anti-apoptotic genes early after TNF stimulation.

Increased TRAF2 degradation impairs activation of NF-κB and MAP kinase signaling. A, B OTUD7b-sufficient and -deficient BMDC were stimulated with 50 ng/ml of TNF. Cells were harvested after 0 (unstimulated), 15, 30, 60 and 120 mins, respectively, and stained for the indicated proteins by WB (A, left panel; B, upper panel). RT-qPCR analysis of relative gene expression of Bcl2l1 and cflar at 120 and 360 min after TNF stimulation (A, top right panel). Mean ± SEM is shown (n = 3 per group and timepoint). In B (lower right panel), relative protein levels of TRAF2 determined by WB analysis and normalized to GAPDH are shown. In A and B, all bars represent mean ratio ± SEM (n = 3 per group and timepoint), (Student’s t-test, *p < 0.05). C OTUD7b-sufficient and -deficient BMDC were either stimulated with 50 ng/ml of TNF or a combination of TNF and MG132 (10 µM) as indicated. Cells were harvested from unstimulated samples and after 30 min of TNF stimulation. Isolated proteins were analyzed as indicated by WB. D–G Protein lysates of unstimulated and TNF stimulated (50 ng) OTUD7b-sufficient and -deficient BMDC were immunoprecipitated with anti-TRAF2 (D: upper panel, E, F: upper panel), anti-OTUD7b (D, F: lower panel) and anti-RIPK1 antibodies (G), respectively, and immunoblotted for the indicated proteins. In (A–G), representative WBs from one of three experiments each are shown.