Ipla2β

Proteins from liver homogenates (30 μg) or ER fractions (10 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Membranes were incubated with a primary antibody overnight at 4 °C. Primary antibodies obtained from Santa Cruz Biotechnology (Heidelberg, Germany) were iPla2β (cat# sc-14463), Xbp-1s (cat# sc-8015), Chop (cat# sc-7351), Srsf3 (serine/arginine-rich splicing factor 3) (cat# sc-13510), Fxr (cat# sc-25309), and calnexin (cat# sc-70481). Other primary antibodies were p-Ire1α (cat# NB100-2323, Novus Biologicals Europe, Abingdon, UK), Scd-1 (cat# ab19862, Abcam, Berlin, Germany), p-eIF2α (cat# 1090-1, Epitomics, Burlingame, CA, USA), Bip (cat# 3177, Cell Signaling, Frankfurt, Germany), p-Perk (cat# 3179, Cell Signaling), Gapdh (cat# 2118, Cell Signaling), and β-actin (cat# A1978, Sigma, Taufkirchen, Germany). After incubation with an HRP-conjugated secondary antibody, blots were developed by using a Luminata Forte ECL reagent (Millipore, Darmstadt, Germany).

Cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, Beijing, China). Samples were then cleared by centrifugation (12,000 rpm, 15 min, 4  °C). Equal amount of proteins (10–20 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The following primary antibodies were used: iPLA₂β (Santa Cruz Biotechnology, Dallas, TX, USA), GRP78 (Santa Cruz Biotechnology, Dallas, TX, USA), calnexin (Abclonal, Wuhan, China), p-IRE1α, ATF6 (Proteintech, Rosemont, IL, USA), ATF4 (Proteintech, Rosemont, IL, USA), p-PERK (Abclonal, Wuhan China), p-eIF2α (Abclonal, Wuhan, China), CHOP, cleaved caspase-3, Bax, p-CaMKII, SERCA2 (Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (MultiSciences, Hangzhou, China), PERK (Abclonal, Wuhan, China), eIF2α (Abclonal, Wuhan, China), CaMKII and GAPDH (MultiSciences, Hangzhou, China). After incubation with the corresponding secondary antibodies conjugated with horseradish peroxidase (HRP) (MultiScience, Hangzhou, China), proteins were detected using a BioRad ChemiDoc MP Imaging system with enhanced chemiluminescence. All other antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).