Fig. 8

LPA increases Traf4-mediated ubiquitination of Smurf2 in SV40 MES13 cells. A SV40 MES13 cells were pretreated with NAC for 1 h and subsequently treated with LPA for 3 h. The level of Smurf2 protein was analyzed by western blotting, quantified using ImageJ software, and normalized to that of β-actin. B and C SV40 MES13 cells were treated with 10 µM LPA for the indicated durations. The mRNA levels of Smurf2 (B) and ubiquitin ligases (C), including Traf4, Trib3, Ttc3, and USP11, were determined by qRT-PCR. D–F SV40 MES13 cells were treated with LPA in the presence or absence of ki16425 for 3 h. D The mRNA levels of Traf4 and Trib3 were determined by qRT-PCR. E The level of Traf4 protein was analyzed by western blotting, quantified using ImageJ software, and normalized to that of β-actin. F The cell lysates were subjected to immunoprecipitation studies by incubating with an anti-Smurf2 antibody followed by blotting with anti-ubiquitin antibody. The protein levels of ubiquitinated-Smurf2 (Ub-Smurf2), ChREBP, and Smurf2 were analyzed by western blotting. Representative images of the blots from three independent experiments are depicted, where the red brackets indicate Ub-Smurf2. G SV40 MES13 cells were transfected with a control (siCon) or Traf4 (siTraf4) siRNA for 6 h, then the medium was replaced with SFM for 16–18 h, and treated with LPA for 3 h. The proteins levels of Traf4 and p-Akt were analyzed by western blotting, quantified using ImageJ software, and normalized to those of β-actin and Akt, respectively. The data are presented as the mean ± SEM of results obtained from four independent experiments. *p < 0.05, **p < 0.01 vs. vehicle only (control) or siCon-BSA; ^#p < 0.05, ^##p < 0.01 vs. LPA only or siCon-LPA