Anti lc3 3868

Anti-LC3 (#3868) and anti-cleaved caspase 3 (#9661) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-SQSTM1 (p62, sc-28359) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-β-actin (A1978) antibody was from Sigma (St. Louis, MO, USA). Osimertinib was purchased from Chemscene LLC. (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO) to 10 mM as a stock solution. Doxazosin was from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and dissolved in DMSO to prepare a 10 mM stock solution. 3-Methyladenine (3-MA) was from Merck Millipore (Darmstadt, Germany) and dissolved in DMSO to 75 mM as a stock solution. Bafilomycin A1 was from Sigma and dissolved in DMSO to 100 µM as a stock solution.

Cell pellets were homogenized in RIPA buffer (Sigma-Aldrich) supplemented with 1% protease and phosphatase inhibitors (Sigma-Aldrich). Samples were kept on ice for 30 min and afterwards centrifuged at 16,000 g for 20 min at 4°C, followed by protein quantification by the bicinchoninic acid assay (Pierce, Thermo Fisher Scientific). Homogenized samples were separated by SDS-PAGE, and transferred to nitrocellulose membranes (Amersham Pharmacia). The membranes were then probed using the following antibodies: anti-Lc3 (3868, Cell Signaling Technology; 1:1000), anti-p62 (5114, Cell Signaling Technology; 1:200), anti-beclin1 (sc-11427, Santa Cruz Biotechnology; 1:500) and anti-α-tubulin (T5168, Sigma-Aldrich; 1:1000). The specificity of antibodies was assessed using positive controls. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) and revealed using a Novex™ ECL Chemiluminescent Substrate Reagent kit (Invitrogen). For the reaction with the endogenous control, membranes were stripped with a stripping buffer (Thermo Fisher Scientific) and re-probed for α-tubulin analysis. Bands were quantified by densitometry using ImageJ.