Hrp coupled anti rabbit immunoglobulin g

Total cell proteins were extracted with RIPA buffer (Teknova) supplemented with protease inhibitor tablet (Pierce). Samples were run on 10% Mini-Protean TGX Gels (BioRad), transferred to PVDF membranes and blocked in 5% nonfat milk (Cell Signaling Technology) for 1 hour. The membranes were incubated with rabbit anti-Gaussia luciferase (Nanolight, Cat#401P, 1:5000) at room temperature for 1 hour. The membranes were washed three times over 20 minutes with PBST (PBS containing 0.1% Tween 20) before being incubated with horseradish peroxidase (HRP) -coupled anti-rabbit immunoglobulin G (Cell Signaling Technology, Cat#7470, 1:10,000) for 45 minutes. After four washes over 30 minutes with PBST, the immunolabeled protein bands were detected by enhanced chemiluminescence (ECL) method (Thermo Scientific). Membranes were then stripped with Restore stripping buffer (Thermo Scientific) and re-probed with rabbit anti-α/β tubulin (Cell Signaling Technology, Cat #2148S, 1:1000) and subsequently HRP-coupled anti-rabbit immunoglobulin G (Cell Signaling Technology, Cat #7470, 1:10,000). For the detection of endogenous and exogenous PNPLA3 in the BA cell line, samples were processed as above, and blots incubated with rabbit anti-PNPLA3 (Abcam, Cat#ab81874, 1:1000) overnight at 4°C and detected as above.