Ab65142

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.

Renal tissues were homogenized in RIPA buffer (P0013, Beyotime, Shanghai, China) supplemented with protease inhibitors (539134, EMD Biosciences Inc., San Diego, CA). After centrifugation (12,000 rpm/min) at 4 °C for 15 min, the supernatant was added to the loading buffer. Protein samples were processed for immunoblot analysis as previously reported (Dixon et al.). The following primary antibodies were used: β-actin (AP0060; Bioworld Technology), protein kinase -like endoplasmic reticulum kinase (PERK, ab65142; Abcam), activating transcription factor 6 (ATF6, ab203119; Abcam), IRE1α (CST3294), p-IRE1α (phosphorylation sites: S724; ab124945; Abcam), JNK (CST9252), p-JNK (phosphorylation sites: Thr183/Tyr185; CST4668), CCAAT-enhancer-binding protein-homologous protein (CHOP, ab179823; Abcam), 4-hydroxynonenal (4-HNE, ab46545; Abcam), glutathione peroxidase 4 (GPX4, ab125066; Abcam). The working concentration of β-actin used was 1:5000, and that of the other antibodies was 1:1000. Secondary antibodies were obtained from Dingguo Co. Ltd. (Beijing, China) and were used at the final dilution of 1:5000. The odesay software was used for analysis (Li Cor, Lincoln, NE).

After treatment, whole cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Solarbio, R0010) at 4°C for 10 min, and the total proteins were quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime, P0010S). The proteins were separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF, Millipore) membranes. After blocking with 5% skimmed milk, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies included monoclonal anti-FLAG M2 (Sigma–Aldrich, F1804, 1:5,000), monoclonal anti-HA tag (Abcam, ab13834, 1:5000), anti-BAX (Abcam, ab53154, 1:5,000), anti-BclXL (ProteinTech, 66020-1-Ig, 1:2,000), anti-Caspase 9 (ProteinTech, 10380-1-AP, 1:1,000), anti-PERK (Abcam, ab65142, 1:500), anti-phosphate eIF2α (Abcam, ab214434, 1:1,000), anti-CHOP (Abcam, ab11419, 1:2,000), anti-Caspase 3 (Abcam, ab208161, 1:1,000), anti-PARP1 (Invitrogen, 436400, 1:2,000) and rabbit polyclonal anti-GAPDH (ProteinTech, 60004-1-Ig, 1:2,000). Following three washes, the membranes were probed with horseradish peroxidase-coupled secondary antibodies. Finally, the membranes were exposed, and signals were recorded using Image Lab software (Bio-Rad) after incubation with enhanced chemiluminescence (ECL) reagents (Beyotime, P0018FM). The relative band intensities were quantified using ImageJ software (V 1.8).