Peroxidase conjugated antibody

The total cellular protein was extracted from PBMCs using a previously described method [12 (link),26 (link)]. Briefly, 40 µg of the isolated protein from PBMC was separated using 7% SDS-PAGE, followed by transfer to the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in a blocking solution overnight at 4 °C, followed by incubation with a primary CCR1 antibody and secondary peroxidase-conjugated antibody (Santa Cruz Biotech, Dallas, TX, USA) at room temperature for 2 h. The CCR1 and β-actin bands were visualised using a Western blotting luminol reagent (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and quantified relative to the β-actin bands. Images were obtained using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

Modified radioimmunoprecipitation (RIPA) buffer was used to extract proteins from the cell. Medium was removed from cells and cells were washed twice with ice-cold PBS before addition of ice-cold RIPA buffer containing 1× Complete Mini EDTA-free protease inhibitor tablet (Roche Diagnostics). Protein concentration of the whole cell lysates was determined using the Bradford assay [12 (link)]. Proteins were separated by SDS-PAGE. Protein bands were then transferred to nitrocellulose paper and incubated with 1/200 diluted AR antibody (N-20) [Santa Cruz Biotechnology] and peroxidase conjugated antibody respectively. Peroxidase linked antibody was purchased from Amersham™ (GE Healthcare Biosciences). β-actin levels were used as a loading control. Protein bands were visualized after chemiluminescent reaction.

Venetoclax and doxorubicin were obtained from LC laboratories (Woburn, MA, USA). Bovine serum albumin and protease cocktail inhibitor reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA). High-Capacity cDNA Reverse Transcription kit and SYBR^® Green PCR Master Mix were obtained from Applied Biosystems^® (Foster City, CA, USA). Primary antibodies to target proteins and peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Chemiluminescence Western blot detection kits, Muse^® Count & Viability Kit, Muse^® Annexin V & Dead Cell Kit, Muse^® Caspase 3/7 Kit, Muse^® MitoPotential Kit, Muse^® Oxidative Stress Kit, and Muse^® Cell Cycle Kit were all obtained from EMD Millipore Co. (Billerica, MA, USA). All other chemicals were obtained from Fisher Scientific Co. (Toronto, ON, Canada).