Cell lysis buffer for western blotting and immunoprecipitation

Total protein was extracted using a cell lysis buffer for Western blotting and immunoprecipitation (Beyotime, Shanghai, China) containing PMSF and a protease inhibitor cocktail. Proteins (30 μg/well) were separated using 10% SDS/PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The primary antibodies used in this study were against anti-HAPSTR1 (1:1000, OTl2B8; Origene), anti-HAPSTR1 (1:500, OTI2D1; Novusbio, Centennial, CO, USA), anti-LRPPRC (1:5000, 21175-1-AP; Proteintech), anti-β-actin (1:1000, 20536-1-AP; Proteintech), anti-Ub (1:1000, 10201-2-AP; Proteintech), anti-N-cadherin (1:5000, 66219-1-Ig; Proteintech), anti-Beclin1 (1:2000, T55092; Abmart, Berkeley Heights, NJ, USA), anti-P62/SQSTM1 (1:2000, T55546; Abmart), anti-ATG5 (1:2000, T55766; Abmart), anti-vimentin (1:500, WL01960; Wanleibio), anti-snail (1:500, WL01863; Wanleibio), and anti-E-Cadherin (1:500, WL01482; Wanleibio).

K-562 cells (5 × 10⁵/well) were plated into 6-well plates. After 5 h, they were treated with 12-epi-napelline (0, 12.5, 25, and 50 μg/ml) for 24 h. HL-60 cells (1 × 10⁶/well) were plated into 6-well plates, incubated for 5 h, and treated with 12-epi-napelline (0, 12.5, 25, and 50 μg/ml) for 24 h. Cells were collected and lysed in 100 μl of cell lysis buffer for western blotting and immunoprecipitation (Beyotime Biotechnology, Shanghai, China) that was supplemented with phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China). Total protein was extracted, and the protein concentration was determined using the Bradford Protein Assay Kit (Tiangen, Beijing, China). Total protein was mixed with an appropriate amount of 5x SDS-PAGE Sample Loading Buffer (Beyotime Biotechnology, Shanghai, China), boiled, separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes, which was blocked with 5% nonfat dry milk in TBST buffer. The primary antibodies were added at proper dilution and incubated at 4°C overnight, and the secondary antibody was added and incubated for 60 min. Finally, the protein was measured by BeyoECL Plus (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's protocol using a gel imaging system (SYSTEM Gel Doc XR+, Bio-Rad, Hercules, CA, USA).