Ab203119

Following the desired treatments, CHO cells were washed and harvested on ice, and then lyzed with ice-cold protein extraction buffer (Beyotime, Nanjing, China). The protein concentration was determined using BCA protein assay kit (ComWin, Beijing, China). Equal amounts of protein were loaded into gel wells, separated by electrophoresis on SDS-polyacrylamide gels and transferred onto PVDF transfer membrane (Millipore, Bedford, MA). The transferred blots were blocked with blocking agents (5% w/v BSA and 0.05% Tween-20 in TBS) for 1 h at room temperature. Blots were then incubated in a certain proportion of specific primary antibodies: anti-GRP78 (ab25192 Rabbit monoclonal, Abcam), anti-ATF-6 (ab203119 Rabbit polyclonal, Abcam), anti-PERK (C33E10 Rabbit polyclonal, Abcam), anti-IRE1 (ab37073 Rabbit polyclonal, Abcam), anti-CHOP (ab11419 Mouse monoclonal, Abcam), anti-Beclin1 (ab55878 Rabbit polyclonal, Abcam), anti-LC3B (ab81785 Rabbit polyclonal, Abcam) (1:1000) overnight at 4°C. These membranes were rinsed 3 times for 10 min each with TBS-Tween (Sigma, St. Louis, MO, USA) and incubated with HRP conjugated secondary antibodies (1:5000) (CWBio, Beijing, China). After washing, protein bands were visualized by an enhanced chemiluminescence detection kit (ComWin, Beijing, China). Each band density was quantified using Image J software (GE Healthcare, Piscataway, New Jersey, USA).

The primary antibodies used are listed as follows: anti-cytochrome c (1:1,000, 10993-1-AP, Proteintech, rosemont, IL, USA), anti-caspase-9 (1:1,000, #9508, Cell Signaling, Danvers, MA, USA), anti-PERK (1:1,000, #3192, Cell Signaling, Danvers, MA, USA), anti-phosphorylated-PERK (anti-p-PERK; Thr982;1:800, DF7576, Affinity Biosciences, Cincinnati, OH, USA), anti-IRE1 (1:500, ab37073, Abcam, Cambridge, MA, USA), anti-p-IRE1 (phosphor S724; 1:1,000, ab48187, Abcam, Cambridge, MA, USA), anti-activating transcription factor 6 (anti-ATF6; 1:1,000, ab203119, Abcam, Cambridge, MA, USA), anti-N’ATF6 (1:800, ab37149, Abcam, Cambridge, MA, USA) and anti-β-actin (1:3,000, GTX109639, GeneTex, Irvine, CA, USA). The ER stress activator Tunicamycin (TM) and the p-PERK inhibitor GSK2656157 were obtained from MedChem Express (Monmouth Junction, NJ, USA). The p-IRE1 inhibitor 3,5-dibromosalicylaldehyde (DBSA) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan).

The OSCC samples were analyzed immunohistochemically using antibodies against PERK (Abcam, Cat No: ab65142), ATF6 (Abcam, Cat No: ab203119), or GRP78 (Abcam, Cat No: ab21685). Next, the brief deparaffinization of 4-μm sections was made in xylene, followed by dehydration in an ethanol diluent serial, and 0.3% hydrogen peroxidase was used to inhibit the activity of endogenous peroxidase at room temperature for 30 min. Then, a microwave oven was adopted to perform antigen retrieval before the blocking of non-specific binding with 5% normal goat serum at 37 °C for 15 min. The overnight incubation of sections was performed with the primary antibody at 4 °C in a humidified box. After three washes with PBS, biotinylated anti-mouse immunoglobulin was adopted for the incubation of sections at 37 °C for 30 min. The visualization of samples was made using 3, 3-diaminobenzidine tetrahydrochloride, followed by counterstaining with hematoxylin. For the analysis, the positive percentages of ER stress-related proteins were explored in five random areas of every sample using Image J.