Western blot for ER stress markers

HET1A cells were treated with bile acids for 6 h. For PP2 pre-treatment studies PP2 (10 uM) was added 24 h prior to co-treatment with bile acids for 6 h. For salubrinal and GSK2606414 pre-treatment studies, these compounds were added 1 h prior to co-treatment with bile acids. Cells were lysed with NP40 buffer and protein quantified using a Pierce^TM BCA kit (Thermo Fisher Scientific Waltham, MA USA). Equal concentrations of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% Marvel, primary antibodies; pPERK/Total PERK/total IRE-1/pSrc/total src/pEFI2a/total EIF2α/tubulin/actin (Cell signalling Technologies Inc. Danvers, MA, USA) or pIRE-1 (Abcam, Cambridge, United Kingdom) were incubated overnight in 5% BSA/TBST. HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc. Dallas, Texas) were incubated for 1 h and detected using Pierce^TM ECL substrate (Thermo Fisher Scientific, Waltham, MA USA).