Xbp 1s

PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) was used to lyse cells or tissues. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore Inc., Billerica, MA). Membranes were blocked with 5% skim milk and incubated with the following antibodies: E-cadherin, vimentin, α-SMA, fibronectin, GRP78, XBP-1s, phosphorylated c-Src, and β-actin (Santa Cruz). The blots were detected after incubation with horseradish peroxidase-conjugated secondary antibodies by an enhanced chemiluminescence detection system (Pierce, Rockford, IL).

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).