Fig. 6

LPA inhibits the ubiquitination of ChREBP by downregulating Smurf2 in SV40 MES13 cells. A SV40 MES13 cells were treated with LPA in the presence or absence of ki16425 for 3 h. B The cells were transfected with a control siRNA (siCon) or Smurf2 siRNA (siSmurf2) for 6 h, then the medium was replaced with SFM for 16–18 h, and subsequently treated with LPA for 3 h. C The cells were transfected with pCMV5B-Flag-Smurf2 wt (Smurf2 wt) or pCMV5B-Flag-Smurf2 C716A (Smurf2 C716A) for 6 h, then the medium was replaced with SFM for 16–18 h, and subsequently treated with LPA for 3 h. (A-C) The cell lysates were subjected to immunoprecipitation studies by incubating with an anti-ChREBP antibody followed by blotting with anti-ubiquitin antibody. The protein levels of ubiquitinated-ChREBP (Ub-ChREBP), 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-ChREBP