Sc 166659

LO2, HepG2, SK-Hep1 cells, or liver tissues were homogenized in immunoprecipitation assay lysis buffer (R0010, Solarbio, Beijing, China). Liver lysates (40 µg) were separated on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following blocking, membranes were probed with mouse monoclonal antibodies against ATF6 (sc-166659, 1:1000, Santa Cruz Biotechnology), eIF2α (sc-133132, 1:1000), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; sc-365062, 1:1000), HNF1α (sc-393668, 1:1000), p-RelA (sc-166748, 1:1000), or RelA (sc-514451, 1:1000), rabbit monoclonal antibodies against ATF4 (11,815, 1:1000, Cell Signaling Technology), cleaved caspase-3 (9664, 1:1000, Cell Signaling Technology), GRP78 (ab108615, 1:10,000, abcam), XBP1s (40,435, 1:1000, Cell Signaling Technology), or p-eIF2α (3398, 1:1000, Cell Signaling Technology), or rabbit polyclonal antibody against caspase-12 (2202, 1:1000, Cell Signaling Technology), protein bands were detected with enhanced chemiluminescent and images were processed using Quantity One software (Bio-Rad, Hercules, CA, USA). Densitometric analysis was used to detect the level of each protein relative to the control.

Cells were detached, washed, and immediately lysed using radio-immunoprecipitation assay buffer (Roche, Basel, Switzerland). Protein concentration was determined by Pierce™ BCA protein assay (Thermo Fisher Scientific). Approximately 20 µg of protein were loaded on 10% SDS polyacrylamide gels for electrophoresis and subsequently blotted on PVDF membranes (Bio-Rad, Hercules, CA, USA). Ponceau S staining was used to validate equal protein load. Membranes were then blocked using 5% non-fat dried milk (NFDM) in TBST for 1 h. Primary antibodies against phospho-eIF2α (1:500 in 5% NFDM, ab32157, Abcam, Cambridge, UK), eIF2α (1:1000 in 5% NFDM, ab5369, Abcam), and ATF6 (1:1000 in 5% NFDM, sc-166659, Santa Cruz) were applied overnight at 4 °C. Following several washing steps with TBST, membranes were incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (1:2000 in TBST, 7074 and 7076, Cell Signaling, Danvers, MA, USA) for 1 h at room temperature. Detection of proteins was realized by addition of Clarity™ Western ECL Substrate (Bio-Rad) and subsequent visualization using a Chemostar chemiluminescence imager (Intas, Goettingen, Germany). Quantitative analysis of signal intensity was performed with LabImage software (version 4.2.3, Kapelan Bio-Imaging GbmH, Leipzig, Germany). Original blots see File S1.