Odysseyxl

Cells were plated in EMEM containing 10% FBS and allowed to attach for 6 h. Cells were then starved for 24 h in EMEM containing 0.1% FBS prior to stimulation with 2ng/mL TGFβ with or without the indicated concentration of compound for 30 min. Total protein was isolated using RIPA buffer (Thermo Scientific, Rockford, IL) and quantified with a BCA protein assay kit (Thermo Scientific, Rockford, IL) according to manufacturer’s instructions. Membranes were probed for phospho-p38 (Cell Signaling, Danvers, MA #9211) and total p38 (Cell Signaling, Danvers, MA #9212) followed by IR conjugated secondary antibodies (Li-Cor Biosciences, Lincoln, NE). Bands were visualized and quantified using a Li-Cor OdysseyXL.

Antibodies used for Western blot were purchased from Abcam (Cambridge, MA): CBS (ab96252; RRID:AB_10678974); Novus Biologicals (Centennial, CO): SQRDL (NBP1-84510; RRID:AB_11027088); Invitrogen (Carlsbad, CA): CSE (PA5-29725; RRID:AB_2547199), and ETHE1 (PA5-56040; RRID:AB_2641135); and Cell Signaling Technology (Danvers, MA): GAPDH (2118; RRID:AB_561053). The Criterion Western Blot system (Bio-Rad) and Li-Cor Odyssey XL were used to measure protein expression. Image Studio software was used to quantify band intensity and blots were normalized to GAPDH. Fold change of the treatment group from the control group was calculated where appropriate.