Anti bax

Cellular proteins were extracted with M-PER™ protein extraction kit (Thermo Fisher Scientific, cat. No. 78501). Proteins were quantified (BCA assay kit; cat. No. 23227; Thermo Fisher Scientific) and 50 μg total proteins were loaded on 10% SDS-PAGE gels. Proteins were then fractionated and transferred to polyvinylidene difluoride (PVDF) membranes (0.45 μm, Merck KGaA), blocked with 5% bovine serum albumin (BSA, AR2440, Sangon Biotech Co., Shanghai, China) for 1 h at RT. Subsequently, the membranes were incubated with primary antibodies at 4˚C overnight, and then incubated with secondary antibodies, horseradish peroxidase-conjugated secondary antibodies against mouse or rabbit (1:1000, Beyotime Institute of Biotechnology, Haimen, China) for 1 hour at RT. Finally, the bands were visualized with the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA). Primary antibodies: anti-EZH2, anti-LC3B, anti-Bax, anti-P62, anti-Bcl-2 (1:1000, HuaAn Biotechnology, Hangzhou, China) and anti-GADPH (1:1000; Hangzhou Goodhere Biotechnology).

Immunohistochemistry staining were performed using Biotin-Streptavidin HRP Detection kit (Zhongshan Bio, Beijing, China) according to the manufacturer's procedure. ⁴³ (link) in brief, tumor tissues obtained from A549, NCI-H460 and NCI-H520 nude mice xenografts were formalin-fixed, paraffin embedded, and then cut into 5-μm sections. After antigen retrieval with autoclaving in citric acid, and inactivating endogenous peroxidase with 3% H₂O₂, the slides were incubated with the rabbit anti-mouse Ki67 monoclonal antibody (Abcam, 1:100 dilution), anti-Bax (HangZhou HuaAn Biotechnology, China, 1:100 dilution), anti-Bcl-2 (Antibody Revolution, 1:100 dilution) and anti-dCK (Biorbyt, 1:100 dilution) antibody overnight at 4°C. Second antibody conjugated with biotin was applied for 20 min at room temperature. To determine the distribution of metuzumab in tumor tissues, the slides were incubated with the biotin labeled goat anti-human antibody (ZSGB-BIO, China. 1: 150). The sections were developed in 3,3-diaminobenzidine(DAB) and counterstained with hematoxylin. The expression level was determined based on the integrated optical density (IOD) using Image Pro Plus 6 Software (Media Cybernetics, USA), according to the method described previously.⁴⁴ (link)

Cells were harvested and lysed with RIPA lysis buffer (Beyotime Biotechnology, China). The total proteins were quantified by Bradford protein assay kit (PIERCE). The proteins were separated by SDS-PAGE and transferred into PVDF membrane. Membranes were blocked in blocking buffer (5% non-fat dry milk/0.1% Tween 20 in TBS) for 1h at room temperature, before being incubated at 4°C with the appropriate antibody in blocking buffer. The membranes were washed and incubated with the appropriated peroxidase conjugated secondary antibody. After washing, the proteins level was detected using ECL reagents (GE Healthcare). The following primary antibodies were used: anti-PCNA (Millipore, 1:250 dilution), anti-Bax (HangZhou HuaAn Biotechnology, China, 1:100 dilution), anti-Bcl-2 (Antibody Revolution, 1:250 dilution), anti-full length caspase-3 (Antibody Revolution, 1:100 dilution), and anti-dCK (Biorbyt, 1:500 dilution). Anti-rabbit (Pierce, 1:6000 dilution) or anti-mouse HRP-conjugated antibodies (Pierce, 1:3000 dilution) were used for secondary antibody reactions.