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. 2017 Sep 5;32(1):230–242. doi: 10.1096/fj.201700415RR

Figure 4.

Figure 4.

USP48 phosphorylation is required for TRAF2 ubiquitination and stability. A) Inhibition of GSK3β promotes TRAF2 degradation. Beas2B cells were treated with or without TWS119 (10 μM) for 1 h; then cells were treated with CHX (20 μg/ml) plus TNF-α (10 ng/ml) for 0 to 8 h. Cell lysates were examined by immunoblotting with TRAF2 and β-actin antibodies. Data are presented as means ± sd (n = 3). *P < 0.01 compared to CHX + TNF-α-treated cells. B) Inhibition of GSK3β promotes TRAF2 ubiquitination. Beas2B cells were treated with TWS119 (10 μM) for 1 h. Denatured cell lysates were subjected to immunoprecipitation with TRAF2 antibody, followed by immunoblotting with ubiquitin antibody. Input lysates were immunoblotted with TRAF2 and β-actin antibodies. C) Overexpression of GSK3βS9A reduces TRAF2 ubiquitination. Beas2B cells were transfected with empty vector or Gsk3β-HA plasmids and incubated for 48 h. Denatured cell lysates were subjected to immunoprecipitation with TRAF2 antibody, followed by immunoblotting with ubiquitin or TRAF2 antibody. Input lysates were immunoblotted with HA, TRAF2, and β-actin antibodies. D) Phosphorylation motif mutant of USP48 loses effect of USP48 on stabilization of TRAF2. Beas2B cells were transfected with Usp48-v5 or Usp48Δ886–890-v5 and incubated for 48 h. Cells were then treated with CHX (20 μg/ml) plus TNF-α (10 ng/ml) for 0 to 8 h. Cell lysates were examined by immunoblotting with TRAF2, V5, and β-actin antibodies (n = 3). *P < 0.01 compared to Usp48-v5-transfected cells. E) Overexpression of GSK3β phosphorylation motif mutant of USP48 increases TRAF2 ubiquitination. Beas2B cells were cotransfected with Usp48-v5 or Usp48Δ886–890-v5 plasmids for 48 h. Denatured cell lysates were subjected to immunoprecipitation with TRAF2 antibody, followed by immunoblotting with ubiquitin antibody. Input lysates were analyzed by immunoblotting with V5, TRAF2, and β-actin antibodies. Shown are representative blots from at least 3 independent experiments. F) Scheme showing GSK3β-mediated USP48 phosphorylation increases TRAF2 stability. Data are presented as means ± sd.