Western Blot Analysis of Signaling Proteins

Western blotting was performed according to a previously described protocol (35 (link)). Briefly, cells in a 6-well plate were lysed on ice with RIPA buffer containing protease inhibitors, and the lysates were centrifuged at 12,000 rpm at 4°C for 10 min. The protein concentration was measured using the BCA Protein Assay Kit (23227; Thermo Fisher Scientific, Waltham, MA, USA). Then proteins were resolved on 10% SDS-PAGE gels and electrotransferred to the Pure Nitrocellulose Blotting Membrane (Pall Corporation, Port Washington, NY, USA). The membrane was blocked in 5% milk, and incubated overnight at 4°C with the following primary antibodies: RAB31 (1:400, 16182-1-AP; Proteintech, Rosemont, IL, USA), GLI1 (1:1,000, ab134906; Abcam, Cambridge, MA, USA), cyclin D1 (1:5,000, ab134175; Abcam), c-Myc (1:1,000, ab39688; Abcam), B-cell lymphoma 2 (Bcl-2) (1:1,000, ab32124, Abcam), and Bcl-2-associated X protein (BAX) (1:1,000, ab32503, Abcam). After washing, the membrane was incubated at room temperature for 1 h with the same secondary antibodies as those used in our previous study (1:3,000; WeiAo Pharmaceutical, Sichuan, China) (35 (link)). The protein signals were detected using the ChemiDoc^TM Imaging System (Bio-Rad, Hercules, CA, USA).