Ab192591

The sections of colon tissue were deparaffinized by xylene, hydrated with gradient ethanol, incubated with 0.1% Tritonx-100 for 30 min, and rinsed 3 times with PBS for 5 min each. The sections were blocked by 5% BSA and 10% sheep serum successively for 30 min. The sections were incubated separately with primary antibody ERK1/2 (ab17942, Abcam), p-ERK (ab192591, Abcam), p38 (ab31828, Abcam), p-p38 (ab47363, Abcam), JNK (ab208035, Abcam), and p-JNK (ab124956, Abcam) in a wet box at 4°C overnight, washed 3 times with PBS for 5 min per time and second antibody at 37°C for 40 minutes, and washed with PBS 3 times for 5 minutes per time. Horseradish peroxidase-labeled streptavidin protein was added dropwise at 37°C for 40 minutes. The sections were rinsed 3 times with PBS for 5 minutes each time, developed by DAB, microscopically observed, and photographed.

The cells were seeded in a 24-well plate (5 × 10⁴ cells/well) with cell climbing slides overnight, and fixed in 4% paraformaldehyde. Immunofluorescence staining was carried on with Rabbit monoclonal anti-p-PERK (p-T982, ab192591, Abcam, 1:80) and mouse monoclonal anti-CHOP antibody (66741-1-Ig, Proteintech, 1:50) and corresponding second antibody with red/green fluorescence light were used, according to the previous method [37 (link)].

Primary cortical neurons or brain tissues were lysed with RIPA buffer containing phosphatase and protease inhibitors. After measurement with BCA kits, the proteins were denatured by boiling for 10 min and then loaded on 8-12% SDS‒PAGE gels, followed by transfer from the gels to activated PVDF membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with the following primary antibodies: anti-PI3K (ab154598, 1:2000), anti-p-Akt (ab38449, 1:1000), anti-Akt (ab283852, 1:2500), anti-GRP78 (ab21685, 1:3000), anti-p-PERK (ab192591, 1:1000), anti-PERK (ab79483, 1:2500), and anti-p-IRE1α (ab124945, 1:2500) were obtained from Abcam, USA and anti-IRE1α (#3294, 1:2500), anti-ATF6 (#65880, 1:2000), anti-CHOP (#5554, 1:2000), anti-Caspase12 (#35965, 1:2500), anti-Caspase3 (#9579, 1:1000), anti-Bcl-2 (#3869S, 1:2000) and anti-Bax (#5023, 1:2500) were supplied by Cell Signaling Technology, U.S.A. Anti-β-actin antibody (GB15004, 1:3000) was purchased from Servicebio Technology (China). After incubation with the primary antibodies overnight, the membranes were washed three times with TBST and incubated with secondary anti-rabbit HRP antibody (GB1213, 1:5000, Servicebio Technology, China). Enhanced chemiluminescence was used to visualize the membranes, and the analysis was performed using ImageJ software.

Total proteins inside cells were extracted utilizing lysis buffer (38733; Sigma‐Aldrich). A total of 30–50 μg protein was separated utilizing 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electronically transferred to polyvinylidene difluoride membranes (IPFL00010; Millipore). Subsequently, immunoblotting was applied utilizing rabbit monoclonal antibodies against MEK2 (ab32517), p‐MEK2 (ab278564), ERK (ab279084), p‐ERK (ab192591), and GAPDH (ab8245'; Abcam). The blots were then visualized utilizing a chemiluminescence detection system (Amersham Pharmacia Biotech).