Xbp1s

In ice-cold lysis buffer (1× phosphate-buffered saline (PBS), 0.1% sodium dodecyl-sulfate (SDS), 0.5% NP-40, and 0.5% sodium deoxycholate) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland), SH-SY5Y cells were lysed and incubated on ice for 30 min. Afterward, we boiled samples with 1× SDS sample buffer for 10 min, separated them by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred them to membranes. With TBST buffer (20 mM Tris-buffered saline and 0.1% Tween-20) containing 5% non-fat milk powder, membranes were incubated for 1 h at room temperature with FLAG (1:1,000; Cat. No. 2368S; Cell Signaling Technology Inc., Danvers, MA, USA), GAPDH (1:5,000; Cat. No. 60004-1-Ig; Proteintech, Rosemont, IL, USA), GRP78 (1:3,000; Cat. No. 11587-1-AP; Proteintech, Rosemont, IL, USA), p-IRE1 (1:500; Cat. No. AF7150; Affinity, West Bridgford, UK), XBP1s (1:2,000; Cat. No. 24868-1-AP; Proteintech, Rosemont, IL, USA), and ATF6 (1:1,000; Cat. No. DF6009; Affinity, West Bridgford, UK) primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Enhanced chemiluminescence reagent (ECL; Cat. No. 170506; Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to visualize the immunoblots.

Antibodies to the phosphorylation of the CCAAT-enhancer-binding protein homologous protein (CHOP), spliced X-box binding protein 1 (XBP1s), and α-subunit of eIF2 (p-eIF2α) were purchased from Affinity Biosciences (Victoria, AU, USA). GAPDH antibody was purchased from Bioworld (Shanghai, China). Anti-rabbit secondary antibody was provided by Signaling Technology (Danvers, MA, USA). All antibodies were used at the manufacturer’s recommended dilutions.