Fc0077

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).

Cells cultured on the glass coverslip were washed with PBS and were fixed with 4% paraformaldehyde. Then, cells on coverslips were permeabilized at 4°C by using 0.25% Triton X‐100 (MP Biomedicals, 9002‐93‐1). Next, coverslips were blocked by 5% heat shock bovine albumin (MP Biomedicals, FC0077). Coverslips were incubated overnight at 4°C with primary antibodies (MAP2). Then, coverslips were incubated with a secondary antibody. Coverslips were fixed on the glass slide and then visualized with a confocal laser scanning microscope (Nikon).