Protein a g kit

Co-IP was performed using a protein A/G kit (Epizyme, Shanghai, China). The beads were activated via overnight incubation in 25 µL of lysis buffer. The suitably treated MCF-7/DDP cells were homogenized in cell lysis buffer at 4 °C for 30 min, and protein concentrations were measured using the BCA assay (Solarbio, Beijing, China). Around 500 µL of each lysate was incubated overnight with the anti-AEG-1 antibody (1 µg, Abcam, ab227981) or with IgG (1 µg, Bioss, Beijing, China, bs-0297R) in the presence of activated beads. The resulting precipitate was washed with lysis buffer, separated via SDS-PAGE and probed using the indicated antibodies with immunoblotting.

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.