Sq201

PROTACs, E3 ligands, MLN8237 (S1133, Selleck), MLN4924 (B1036‐5.1, APExBIO), and Bortezomib (T2399, TargetMol) treated samples as well as released synchronized cells were subjected for immunoblot analysis. Whole cell lysates were extracted using standard Western blot procedures. Fresh cells were lysed on ice in an RIPA buffer (50 × 10⁻³m Tris pH = 8.0, 1 × 10⁻³m EDTA, 150 × 10⁻³m NaCl, 1% NP‐40, 0.5% sodium deoxycholic acid, 0.1% SDS) containing phosphatase and protease inhibitors (C0002 and C0001, TargetMol) for 15 min. Ultrasound was performed at 80 Hz for 5 min and the impurities were removed by centrifuge at 12 000 RPM for 10 min. The concentration of protein supernatant was determined by the Bradford method and an equal amount of protein was subjected for electrophoresis using the procedure of 60V for 30 min followed by 120 V for 1 h. The protein was transferred onto a PVDF membrane with 300 mA for 90 min. After being blocked at room temperature for 1 h with 5% bovine serum albumin (BSA, FC0077, MP), the membrane was incubated overnight with the indicated antibodies at 4 °C. Subsequently, the PVDF membrane was washed three times with TBST and incubated with the peroxidase‐conjugated secondary antibody for 1 h at room temperature. Protein expression was monitored by the BioRad chemiluminescent imaging system with an enhanced chemiluminescence reagent (SQ201, EpiZyme).

RIPA buffer (P0013B, Beyotime, Shanghai, China) was used for protein extraction. After the total protein concentration was determined by a bicinchoninic acid protein assay kit (BCA1, Sigma Aldrich, St. Louis, MO). 20 μg protein samples were separated by 7.5% SDS polyacrylamide gels and transferred onto PVDF membranes (ISEQ00010, Millipore, Massachusetts, United States). The membrane was blocked with 5% non-fat milk in TBST for 1 h and incubated with the indicated antibody at 4°C overnight. Then HRP-conjugated secondary anti bodies (1:5000) were added. Bands were subsequently visualized using the enhanced plus chemiluminescence assay (SQ201, Epizyme, Shanghai, China). Measurement of the bands was conducted on an ImageQuant LAS 4000 mini.

According to the previous method, we extracted proteins from cells and heart tissues for Western blot experiments (Jiao et al., 2022 (link)). In short, we use 7.5–12.5% SDS-PAGE gel for protein separation and transfer it to PVDF membranes. After blocking with 5% nonfat dry milk at room temperature for at least 1 h, the membranes were washed three times with TBST and incubated with primary antibodies overnight in a 4°C refrigerator. The corresponding primary antibodies are as follows: GAPDH (Abcam, ab181602), PTGS2 (Abclonal, A1253), AKT (Proteintech, 10176-2-AP), Phospho-Akt (Abclonal, AP1259), Phospho-mTOR (CST, 5536T), mTOR (CST, 2983T). After collecting the primary antibody, the membranes were reacted with HRP-conjugated secondary antibodies (Beyotime, A0208) for 1 h. At last, the immune blots were observed by enhanced chemiluminescence ECL (Epizyme, SQ201).