AML12 cells (ATCC CRL-2254) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone. Hep3B cells were cultured in MEM supplemented with 1% Non-Essential Amino Acids (NEAA) and 10% FBS. Smurf1KO MEFs were cultured in DMEM supplemented with 10% FBS. Primary hepatocytes were isolated by a two-step collagenase perfusion of the liver and cultured as described [45 (
link)]. Flag-tagged PPARγ1, PPARγ2 plasmids, and PPRE-Luc reporter plasmids were obtained from Addgene. Flag-tagged PPARγ2ΔPY plasmid was generated using
Site Directed Mutagenesis Kit (Agilent Technologies). Myc-tagged Smurf1, Smurf2, and Smurf1CA mutant, HA-tagged Ubiquitin plasmids were described before [13 (
link), 18 (
link), 46 (
link)].
Anti-Smurf1 (Novus, 1D7);
anti-Smurf2 (Abcam, EP629Y3);
anti-PPARγ (Santa Cruz, sc-7273); anti-PPARα (Rockland, 600-401-4215);
anti-PPARδ (ThermoFisher, PA1-823A);
anti-HSC70 (Santa Cruz, B-6);
Anti-Flag-Peroxidase (A8592, Sigma);
anti-HA (Covance, HA11); and
anti-Myc (Santa Cruz, 9E10) were used for western blotting and immunoprecipitation. Knockdown experiments were performed using the following siRNAs:
siPPARγ (J-040712-05 and J-040712-07, Dharmacon). Validated siSmurf1, siSmurf2 and siNS were previously described [47 (
link), 48 (
link)].
Zhu K., Tang Y., Xu X., Dang H., Tang L.Y., Wang X., Wang X.W, & Zhang Y.E. (2018). Non-proteolytic ubiquitin modification of PPARγ by Smurf1 protects the liver from steatosis. PLoS Biology, 16(12), e3000091.
