Enhanced chemiluminiscence method

Cell or tissue lysates were lysed in 4% SDS buffer followed by needle homogenization. Equal amounts of protein (15–40 μg) were mixed with loading dye, boiled for 8 min, separated on a denaturing SDS–PAGE gel and transferred to a PVDF membrane. The membrane was blocked in 5% milk/TBS/0.1% Tween for 1 h and incubated with antibodies against pERK, ERK, Vimentin, (Cell Signaling, Danvers, MA, USA), αSMA (abcam), β-Actin (Santa Cruz Biotech, Santa Cruz, CA, USA). The membrane was washed with TBS-0.1% Tween and then incubated with HRP conjugated secondary antibody (Santa Cruz Biotech) at room temperature for 1 h and rewashed. Protein bands were visualized by an enhanced chemiluminiscence method (Thermo, Waltham, MA, USA) and resolved digitally per the manufacturer’s specifications. For RAS activation assay, a standard kit was purchased and used per manufacturer specification (Thermo). All experiments were performed in triplicate unless otherwise specified.

Cell or tissue lysates were lysed in 4% SDS buffer followed by needle homogenization. Equal amounts of protein (15-40 μg) were mixed with loading dye, boiled for 8 min, separated on a denaturing SDS-polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked in 5% milk/TBS/0.1% Tween for 1h and incubated with antibodies against ADAM17, NFκB, p21, GAPDH (Santa Cruz Biotechnology, Dallas, TX), Collagen IA (Southern Biotech, Birmingham, AL), TGFβ1 (abcam, Cambridge, MA), pSTAT3, pERK, pAKT (Cell Signaling, Danvers, MA), or ATGL (eBioSci, San Diego, CA).
The membrane was washed with TBS-0.1% Tween and then incubated with HRP-conjugated secondary antibody (Santa Cruz) at room temperature for 1h and rewashed. Protein bands were visualized by an enhanced chemiluminiscence method (Thermo, Waltham, MA) and resolved digitally per the manufacturer's specifications.