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. 2024 Jun 27;15:5441. doi: 10.1038/s41467-024-49854-1

Fig. 5. OTUD5 negatively regulates TAK1 activation and inflammation in podocytes.

Fig. 5

MPC5 cells transfected with Flag-OTUD5 (a) or si-OTUD5 (b) were stimulated with HG/PA for 30 min. Representative western blot analysis of P-TAK1. (n = 3 independent experiments). c, d Representative western blot analysis of P-TAK1 in kidney tissues of each group. (n = 6 samples). MPC5 cells transfected with Flag-OTUD5 (e) or si-OTUD5 (f) were stimulated with HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of ERK, P38, and JNK. (n = 3 independent experiments). MPC5 cells transfected with si-OTUD5 were pretreated with 10 μM Takinib (TAK1 inhibitor) for 1 h before exposure to HG/PA. g Levels of P-TAK1, P-ERK, P-P38, and P-JNK were detected by western blot. h Real-time qPCR showing mRNA levels of Il6 and Tnfα. (n = 3 independent experiments; P values were determined by one-way ANOVA with Bonferroni’s correction and data are presented as mean ± SD). i His-TAK1 was transfected into NIH/3T3 with or without Flag-OTUD5 (WT or C224A). Co-IP was performed with an anti-His antibody, followed by a western blot of TAK1 and TAB2. (n = 3 independent experiments). j MPC5 cells transfected with Flag-OTUD5 (WT or C224A) were stimulated with HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of TAK1, ERK, P38, and JNK. (n = 3 independent experiments). k His-TAK1(WT or K158R) was transfected into NIH/3T3 with or without Flag-OTUD5. Co-IP was performed with an anti-His antibody, followed by a western blot of TAK1 and TAB2. (n = 3 independent experiments). l His-TAK1 (WT or K158R) and Flag-OTUD5 were transfected into MPC5 cells for 24 h and then stimulated by HG/PA for 30 min. Representative western blot analysis of phosphorylated and total protein levels of TAK1, ERK, P38, and JNK. (n = 3 independent experiments).