Anti phospho ire1α

Cannabidiol and VAS2870 were purchased from Sigma (St. Louis, MO, USA). TRAIL and anti-DR5 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-Bak, anti-Bcl-2, anti-Mcl-1, anti-Bcl-xL, and anti-DR4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-BIM, anti-survivin, anti-Bid, anti-IRE1α, anti-phospho-IRE1α, anti-Bip, anti-GRP94, anti-ATF6, anti-eIF2α, anti-phospho-eIF2α, anti-CHOP, anti-cleaved PARP, anti-caspase-3, and anti-caspase-9 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). For the secondary antibodies, anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG HRP were purchased from Cell Signaling Technology.

A total of 25 μg of protein samples were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the gel were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) solution at room temperature for 1 hour and then incubated in the appropriate primary antibodies (diluted in 5% milk in TBST) at 4°C overnight. After fully washing for approximately 30 minutes with TBST, the membranes were incubated in a secondary antibody (diluted in 5% milk in TBST) at room temperature for 90 min. The primary antibody information is as follows: anti-GRP78 (Abcam, Cambridge, MA 1:2000), anti-phospho-IRE1α (Cell signaling, MA 1:1000) and anti-phospho-EIF2α (Cell signaling, MA 1:1000), and anti-ATF6 (Abcam, Cambridge, MA 1:1000). Peroxidase-based detection was performed using chemiluminescence reagent (NEN Life Science). The same membrane was stripped and re-probed using an anti-β-actin antibody (Abcam 1:10000) as the loading control. Densitometric analyses of the Western blots were performed using ImageJ software. We repeated each experiment three times.

Korean Red Ginseng powder was kindly provided by the Korea Ginseng Corporation (Daejeon, Korea). The KRG powders were dissolved in distilled water. N-acetyl-l-cysteine (NAC) and Anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase-3, anti-caspase-9, anti-cleaved PARP, anti-BIM, anti-Noxa, anti-survivin, anti-IRE1α, anti-phospho IRE1α, anti-GRP94, anti-eIF2α, anti-phospho eIF2α, anti-ATF6, anti-PERK, anti-phospho PERK, anti-Bip anti-XBP1s, anti-ATF4, anti-SOD2, anti-SOD1, anti-catalase, anti-NOX4, and anti-NOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bak, anti-BAX, anti-Bcl-2, anti-SOD3, and anti-CHOP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-mouse and Rabbit IgG HRP, the secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA).