Ab11419

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).

HUVECs were lyzed using RIPA lysis buffer (Beyotime, Shanghai, China) and cell supernatants were collected by centrifugation. The protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were isolated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies against Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab243140, 1:500), Cleaved caspase-3 (Abcam, ab2302, 1:500), Caspase-3 (Abcam, ab184787, 1:2000), Beclin-1 (Abcam, ab207612, 1:2000), Atg5 (Abcam, ab108327, 1:10,000), p62 (Abcam, ab207305, 1:1000), GRP78 (Abcam, ab108615, 1:10,000), CHOP (Abcam, ab11419, 1:1000), Caspase-12 (Abcam, ab62484, 1:2000), XBP-1 (Abcam, ab220783, 1:1000) and GAPDH (Abcam, ab32124, 1:1000) overnight at 4°C. Membranes were washed in TBST, followed by incubation with secondary antibodies (Abcam, ab205718, 1:50,000; Abcam, ab205719, 1:20,000) for 1 h at room temperature. Protein bots were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China).

Total proteins were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the concentration of the protein samples was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime). Proteins were subjected to SDS-PAGE and then electrophoretically transferred onto PVDF membranes. Membranes were incubated with primary antibodies overnight at 4°C after blocking in 5% nonfat milk in TBST buffer, which was then coincubated with horseradish peroxidase (HRP)-conjugated anti-mouse/anti-rabbit IgG (Thermo Fisher Scientific). Proteins were visualized using a detection system of enhanced chemiluminescence (ECL), and the bands were analyzed using BandScan ImageJ software. The primary antibodies used in this study were as follows: anti-CHOP (diluted 1 : 1000, ab11419, Abcam), anti-caspase 12 (diluted 1 : 1,000, ab62484, Abcam), anti-GRP78 (diluted 1 : 100, ab21685, Abcam), anti-NLRP3 (diluted 1 : 500, ab214185, Abcam), anti-ASC (diluted 1 : 1000, ab155970, Abcam), anti-pro-IL-1β (diluted 1 : 500, ab2105, Abcam), anti-IL-1β (diluted 1 : 1500, ab9722, Abcam), anti-p-NF-κB (p-p65) (diluted 1 : 2000, ab86299, Abcam), anti-NF-kB p65 (diluted 1 : 300, ab19870, Abcam), anti-TLR4 (diluted 1 : 500, ab13556, Abcam), anti-MyD88 (diluted 1 : 1000, ab133739, Abcam), and anti-β-actin (diluted 1 : 1000, ab8226, Abcam). β-Actin was used as an internal control.

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).

The treated human LO-2 hepatocytes were lysed to isolate proteins, which were subsequently loaded and separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane and incubated with 5% nonfat milk. The membrane was then incubated with primary antibody against NOX-4 (1:1000, ab133303, Abcam), NLRP3 (1:1000, ab263899, Abcam), p-eIF-2α (1:1000, ab169528, Abcam), ATF4 (1:1000, ab184909, Abcam), GADD34 (1:1000, ab236516, Abcam), CHOP (1:1000, ab11419, Abcam), NF-κB p65 (1:1000, ab288751, Abcam) or β-actin (1:1000, ab8226, Abcam), which was further incubated with an HRP-conjugated secondary antibody. Finally, the blots were developed with electrochemiluminescence (ECL) reagents and analyzed using Image J software [25 (link)].