Anti p ire1α

DHA (purity ≥ 98%), Oil Red O, tunicamycin, insulin, dexamethasone, rat-tail collagen and type IV collagenase were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Briefly, the stock solution of DHA was dissolved in 95% ethanol at a concentration of 200 mM, and the working solution was prepared by adding the stock to the culture medium to achieve a final concentration of 25 μM. Epidermal growth factor was a product from Peprotech (Peprotech, Rocky Hill, NJ, USA). The primary antibodies applied in this study were anti-GRP78 (Cell Signaling Technology, Danvers, MA, USA), anti-ACC (Cell Signaling Technology, Danvers, MA, USA), anti-IRE (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ACOX1 (Abcam, Cambridge, UK), anti-p-IRE1α (Abcam, Cambridge, UK) and anti-β-actin (Beyotime, Shanghai, China).

Cell or tissue lysates were extracted in RIPA buffer (Beyotime, Cat# P0013B) Protein concentration was evaluated by bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Cat# 23227) and then analyzed by SDS–PAGE gel electrophoresis and blotting onto PVDF membranes. Primary antibodies used included: anti-ERO1L (Abcam, Cat# ab177156), anti-IRE1α (Abcam, Cat# ab37073), anti-pIRE1α (Abcam, Cat# ab48187), anti-XBP1s (Cell Signaling Technology, Cat# 12782), anti-PERK (Abcam, Cat# ab229912), anti-pPERK (Cell Signaling Technology, Cat# 3179), anti-eIF2α (Cell Signaling Technology, Cat# 9722), anti-peIF2α (Cell Signaling Technology, Cat# 9721), anti-ATF4 (Cell Signaling Technology, Cat# 11815), anti-ATF6α (Santa Cruz, Cat# sc-166659), anti-CHOP (Santa Cruz, Cat# sc-7351), and anti-β-Actin. Primary antibodies were applied in 5% non-fatty milk or primary antibody dilution (Beyotime, Cat# P0256) in TBST and incubated overnight at 4°C, followed by HRP-conjugated secondary antibodies incubation at room temperature for 2 h. Secondary antibodies used included: Goat anti-Rabbit HRP-conjugated IgG (Abcam, Cat# ab6721), Rabbit anti-Mouse HRP-conjugated IgG (Abcam, Cat# ab6728). Images were collected by Amersham Imager 600 (General Electric).

Lysates of CL tissue from each stage were prepared in PRO-PREP protein lysis buffer (iNtRON, Daejeon, Korea). The protein concentration for each sample was estimated using a Bradford dye-binding assay with bovine serum albumin (BSA) as a standard (Bradford, 1976). Aliquots of the proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels. After electrophoresis, the separated proteins were transferred onto nitrocellulose (NC) membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 at 4 °C under mild agitation, the membranes were incubated with 1:2000 diluted anti-CHOP, anti-p90ATF6, anti-CREB-2, anti-3β-HSD (Santa Cruz, Biotechnology, CA, USA), anti-GRP78/BIP, anti-p-eIF2α, anti-IRE1, anti-eIF2a, anti-JNK, anti-p-SAPK/JNK, anti-caspase-3, anti- β-tubulin (Cell Signaling, Beverly, MA, USA), anti-p50ATF6 (IMG, San Diego, CA, USA), and anti-p-IRE1α (Abcam, Cambridge, MA, USA) antibodies. The membranes were then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-goat/mouse/rabbit IgG (Thermo, Rockford, IL). Antibody binding was detected using an enhanced chemiluminescence (ECL) kit (Advansta, CA, USA). Band intensities were quantified using the Image J software (NIH, MD, USA).