Anti pnpla3

Cells were washed twice with precooled PBS, and then the total cellular protein was extracted by adding RIPA lysate containing a protease inhibitor, and protein concentration was determined by the BCA method. A volume of 10 ul (4 ug/ul) protein was taken for gel electrophoresis, and the protein was transferred to the PVDF membrane. After being closed with 5% skimmed milk for 1 hour at room temperature, the primary antibody was incubated overnight, including anti-PNPLA3 (ab81874; Abcam), anticleaved caspase3 (#9664, Cell Signaling Technology), anti-BIP (#3183, Cell Signaling Technology), anti-PERK (#5683, Cell Signaling Technology), anti-p-PERK (Thr981) (sc-32577, Santa Cruz), anti-eIF-2a (#9722, Cell Signaling Technology), anti-p-eIF-2a (Ser51) (#9721, Cell Signaling Technology), anti-CHOP (#2895, Cell Signaling Technology), anti-PUMA (#4976, Cell Signaling Technology), anti-BAX (#5023, Cell Signaling Technology), and anti-β-actin (#4970, Cell Signaling Technology). The membrane was then washed with TBST and incubated with fluorescently labeled secondary antibodies for 1 hour at room temperature. Protein bands were visualized with an infrared fluorescence scanning imaging system (Odyssey ® DLx Imaging System, LI-COR Biosciences). Each experiment was repeated a minimum of three times.

Approximately, 500.000 cells per condition were collected in RIPA buffer (Radio immune precipitation assay buffer, 0.01 mol/L Tris-Cl (pH 8.0), 0.001 mol/L EDTA, 5 × 10⁻⁴ mol/L EGTA, 0.1% sodium deoxycholate, 0.1% SDS, 1% NP-40) and protein concentration was measured using 660 nm protein assay kit. Twenty micrograms per sample were loaded on a SDS-PAGE using 10% polyacrylamide gels. α-SMA was identified using a 1:2000 dilution of the monoclonal mouse anti-humanα-SMA (Sigma-Aldrich), AQP3 was detected using a 1:500 dilution of monoclonal rabbit anti-AQP3 as performed before^{47 (link)}. PNPLA3 was detected with a 1:1000 dilution of the rabbit polyclonal anti-PNPLA3 (Abcam, Cambridge, UK). Band intensity achieved from these antibodies was normalized to band intensity of Calnexin using mouse anti-calnexin 1:3000 (Santa Cruz Biotechnology Inc, Dallas, TX, USA).