Phosphatase inhibitor cocktail

ADSCs were pretreated with PD98059 (HY-12028, 80 μM, MedChemExpress), GsMTx4 (HY-P1410A, 5 μM, MedChemExpress), and Yoda1 (HY-18723, 30 μM, MedChemExpress) for 1 hour as requested, and the cells were harvested 2 hours after LIPUS stimulation without specification. ADSCs were lysed on ice with RIPA buffer (Beyotime, Shanghai, China) containing the protease inhibitor PMSF (Beyotime, Shanghai, China) and Phosphatase Inhibitor Cocktail (YEASEN, Shanghai, China). The protein concentrations of cell lysates were measured using a BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (15 μg) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 10% bovine serum albumin (BSA) for 1 hour at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study were ERK (ab184699, 1 : 10000, Abcam, USA), p-ERK (ab76299, 1 : 5000, Abcam, USA), VEGFA (ab214424, 1 : 1000, Abcam, USA), and GAPDH (ab181602, 1 : 10000, Abcam, USA). After being rinsed with TBST, the membranes were hybridized with secondary antibodies and reacted with ECL solution (NCM Biotech Co., Ltd, Suzhou, China). The chemiluminescent images were taken, and the density of each protein band was determined using ImageJ software.

HEK-293T or HeLa cells were transfected with plasmids, as indicated in the figures, and cultured for 24 hours before collecting the protein lysate. All cells were washed with precooled PBS before collection and lysed in cell lysis buffer for western blotting and IP (Beyotime, P0013) supplemented with a 1% protease inhibitor cocktail (MedChemExpress, HY-K0010) and a 1% phosphatase inhibitor cocktail (Yeasen, 20109ES05) for up to 30 min at 4°C, after which they were centrifuged at 12,000 g for 10 min. Rat cerebral cortex and spleen samples were homogenized in cell lysis buffer with an electric homogenizer, lysed for 1 h at 4°C, and centrifuged at 12,000 g for 10 min at 4°C. The collected supernatants were used for IP using a previously described protocol 40 (link).
The antibodies used for IP were as follows: anti-eIF2α (Proteintech, 11170-1-AP), anti-phospho-eIF2α (Ser51) (Cell Signaling Technology, 3398), and anti-DDDDK (Flag)-tag (Abclonal, AE092).

For co-IP assay, HEK-293T cells were harvested with precooled RIPA lysis buffer (Beyotime, China) containing protease inhibitor PMSF (Beyotime, China) and phosphatase inhibitor cocktail (YEASEN, China). After centrifugation at 14,000×g for 10 min at 4°C, the supernatant was immunoprecipitated against anti-Flag Affinity Gel for 4 h at 4°C. Then, the beads were washed with 1×TBS for three times before boiling with 1 × SDS-PAGE loading buffer (YEASEN, China) to elute the immunoprecipitate. Precipitated proteins were analyzed by SDS-PAGE gel electrophoresis and immunoblotting.