Monoclonal anti bcl 2

Hesperetin (Cat. No. 520-33-2) was purchased from ChemFaces Company (ChemFaces, Wuhan, China) and a 100 mM stock solution was prepared in DMSO (0.1% v/v final concentration) and stored at −20 °C. 3-Methyladenine (3-MA, an autophagy inhibitor) was purchased from Sigma Chemical Co. (St. Louis, MO, USA), and bafilomycin A1 (Baf-A1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-p-AMPKα (Thr172; #2535), polyclonal anti-AMPKα (D63G4) (#5832), polyclonal anti-p-mTOR (Ser2448; #5536), polyclonal anti-mTOR (7C10) (#2983), polyclonal anti-cleaved PARP (#9532), polyclonal anti-Beclin-1 (#3495), polyclonal anti-SQSTM1/p62 (#5114), polyclonal anti-p-Akt (Ser4723; #4060), polyclonal anti-p-Akt (#9272), polyclonal anti-Atg5 (#2630), monoclonal anti-Bcl-2 (#15071), monoclonal anti-Bax (#5023), and monoclonal anti-GAPDH (#0411) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). We also purchased polyclonal-anti-LC-3B (#L7543; Sigma, UK), goat anti-rabbit IgG secondary antibodies (#NA934; GE Healthcare Life Sciences, Chalfont, UK), and IgG HRP-conjugated secondary antibody (polyclonal anti-mouse; #115-035; 1:5000; Jackson ImmunoResearch Laboratories, UK).

The U937 cell line was lysed in 1× RIPA buffer (N653, Amresco, Solon, OH, USA) with 10% proteasome inhibitor. All cell extracts were cleared at 13,000 rpm for 30 min in a microcentrifuge at 4 °C. Proteins were separated using 10% SDS-PAGE and were transferred to a PVDF membrane. The membrane was blocked in 5% nonfat dry milk in PBS buffer with Tween 20. Immunostaining was performed using the following antibodies: monoclonal anti-p-AMPKα (Thr172; #2535), monoclonal anti-AMPKα (#5832), polyclonal anti-p-mTOR (Ser2448; #5536), polyclonal anti-mTOR (7C10) (#2983), polyclonal anti-cleaved PARP (#9532), polyclonal anti-Beclin-1 (#3495), polyclonal anti-SQSTM1/p62 (#5114), polyclonal anti-p-Akt (Ser4723; #4060), polyclonal anti-p-Akt (#9272), polyclonal anti-Atg5 (#2630), monoclonal anti-Bcl-2 (#15071), monoclonal anti-Bax (#5023), and monoclonal anti-GAPDH (#0411) obtained from Cell Signaling Technology; and polyclonal-anti-LC-3B (#L7543; Sigma). The secondary antibodies were goat anti-rabbit IgG (#NA934, GE Healthcare Life Sciences) and IgG HRP-conjugated secondary antibodies (polyclonal anti-mouse; #115-035; 1:5000; Jackson ImmunoResearch Laboratories, London, UK). An ECL Western blotting reagent (GE Healthcare Life Sciences, Massachusetts, MA, USA) was used for protein detection.