Horseradish peroxidase conjugated secondary antibodies

Cells were washed once with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing 1% protease inhibitors (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Protein concentration was subsequently measured with a BCA kit (Thermo Fisher Scientific, Inc.), ~10 µg protein was separated with 8–12% SDS-PAGE gel and blotted onto polyvinylidene fluoride or polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany). Membranes were subsequently blocked with 5% milk at room temperature for 1 h and incubated with primary antibodies against caspase 3 (1:600; cat. no. 9665), caspase 8 (1:1,000; cat. no. 4790), caspase 9 (1:800; cat. no. 9508), p53 (1:1,200; cat. no. 2524), Cyto C (1:1,000; cat. no. 4280), Bcl-2 (1:1,200; cat. no. 3498), Bcl-xL (1:1,000; cat. no. 2762) and GAPDH (1:5,000; cat. no. 2118) (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight, followed by 3 washes with tris-buffered saline with tween and a 1 h incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature (OriGene Technologies Inc., Beijing, China). Protein bands were visualized with an enhanced chemiluminescence kit (Merck KGaA). The density of bands was quantified by ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).

Fetal bovine serum (FBS), RPMI-1640 medium and penicillin/streptomycin were purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). LRP, phosphorylated-glycoprotein (p-gp), MRP1, GAPDH, Ki-67 nuclear antigen and proliferating cell nuclear antigen (PCNA) primary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from OriGene Technologies, Inc. (Beijing, China). DOX and SML were obtained from the Department of Neurosurgery at The Second Affiliated Hospital of Chongqing Medical University (Chongqing, China).

The following primary antibodies were obtained for the present study: p62/SQSTM1 (cat. no. 8025S; Cell Signaling Technology, Inc., Danvers, MA, USA); light-chain 3 (LC3)B (cat. no. L7543); and β-actin (cat. no. A1987) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Horseradish peroxidase-conjugated secondary antibodies were purchased from OriGene Technologies, Inc., (cat. nos. TA-130003 and TA140003, respectively; Rockville, MD, USA) and chloroquine (CQ) diphosphate salt was purchased from Sigma-Aldrich (cat. no. C6628; Merck KGaA).