Anti grp78 antibody

Fresh uterine tissue was fixed in 4% paraformaldehyde, embedded in conventional paraffin, and sliced at 3–8 μm. After dehydration, the sections were placed in citrate buffer and heated to extract antigens. Inactivated endogenous peroxidase was added to the sections in hydrogen peroxide/methanol for 1 h followed by washing in PBS. The sections were incubated in 10% pre-immune serum (Maixin-Biotech, Fuzhou, China) for 30 min at 37 °C prior to incubation with anti-GRP78 antibody (Proteintech) for 12 h at 4 °C. Preimmune serum was used as a negative control. After washing, the sections were incubated with biotinylated antibiotic-rabbit IgG antibody (Maixin-Biotech, Fuzhou, China) for 30 min at 37 °C. Streptavidin labeled with horseradish peroxidase was added with incubation at 37 °C for 30 min. Microscopy (Nikon, Tokyo, Japan) was used to evaluate the sections after staining with DAB chromogenic solution (Maixin-Biotech, Fuzhou, China).

The protein was extracted from fresh-frozen hippocampal tissue using RIPA lysate. The BCA method was used to measure protein concentration. The protein solution and SDS-PAGE loading buffer (5x) were mixed at 1 : 4, then heated at 100°C for 10 min to denature the protein. After this production, equal amounts of proteins were separated using SDS-PAGE gel and transferred onto the PVDF membrane. Then, PVDF membranes were blocked with 10% skim milk and incubated with anti-GRP78 antibody (1 : 5000, Proteintech, China), anti-ATF4 antibody (1 : 2000, Proteintech, China), anti-Bax antibody (1: 5000, Proteintech, China), anti-Bcl2 antibody (1 : 2000, Immunoway, China), anti-CHOP antibody (1 : 1000, Proteintech, China), caspase9 (1 : 1000, CST, USA), TNF-α (1 : 1000, Proteintech, China), IL-6 (1 : 1000, Proteintech, China), IL-10 (1 : 1000, Proteintech, China), and anti-β-actin antibody (1 : 10000, Santa Cruz, USA) overnight at 4°C. Afterwards, the bolts were incubated for 1 hour at 37°C using the corresponding anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (1 : 10000, Proteintech, China). Finally, the protein bands were identified using the ECL Chemiluminescence Kits (Millipore, USA). Protein levels were normalized to β-actin as reference. The relative density of the protein level was quantitated by the ImageJ software.

The protein A/G kit (Epizyme, Shanghai, China) was utilized for conducting Co-IP. The beads were activated after incubated in the lysis buffer (25 µL) overnight. After the appropriate treatment, the cells were subjected to homogenization in a meticulously prepared cell lysis buffer and kept at a consistent temperature of 4 °C for a duration of 30 min. The bicinchoninic acid (BCA) assay (Vazyme Corporation, Nanjing, China) was employed to quantify protein concentrations. Each lysate (approximately 500 µL) was subjected to an overnight incubation with either 4 µg of anti-GRP78 antibody (Proteintech, Cat No. 66574-1-Ig) or 1 µg of IgG (Bioss, Beijing, China, bs-0297R), along with the activated beads. Finally, the precipitate was treated with a lysis buffer. After the elution procedure, the IP was identified through Western blotting (WB) analysis.