Beta actin monoclonal antibody

Nitazoxanide (NTZ) and the cell cycle checkpoint kinase 1 (Chk1) inhibitors MK-8776 and Prexasertib HCl were purchased from Selleck Chemicals (Selleck, Houston, TX, USA). The anti-VP2 monoclonal antibody was purchased from Ingenasa (Ingenasa, Madrid, Spain). Beta-actin monoclonal antibody was purchased from Thermo Fisher (Thermo Fisher, Waltham, MA, USA). The ProteinExt^® Mammalian Total Protein Extraction Kit was purchased from TransGen (TransGen Biotech, Beijing, China). Total RNA extraction was conducted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). The reverse transcription kit FastKing RT Kit (With gDNase) and quantitative PCR kit SuperReal PreMix Plus (SYBR Green) were purchased from TIANGEN (TIANGEN, Beijing, China).

The MCF-7 and MDA-MB-231 cells following 24 h treatment with AKBA, ABA or BA were trypsinized and lysed with RIPA buffer and protease inhibitors (ThermoFisher Scientific). The protein concentrations were checked by Pierce™ Rapid Gold BCA Protein Assay Kit (ThermoFisher Scientific). The lysates were incorporated with Laemmli sample buffer. Western blot analysis of P53 and BCL2 was achieved by loading 50 μg of total proteins and were run by 10% acrylamide gel (Bolt Bis–Tris Plus gels) (Invitrogen, USA) at a constant voltage of 200 V. Following SDS-PAGE, the proteins from the gel were transferred to Nitrocellulose membranes by Pierce power blotter (ThermoFisher Scientific) with a high M.W. pre-programmed approach. The membranes were later blocked with BSA dissolved in TBS/Tween-20 (5% BSA, 0.5% Tween-20 for 1 h), accompanied by immunoblotting with P53 monoclonal antibody (1/1500 dilution) and BCL2 monoclonal antibody (1/50 dilution) (ThermoFisher Scientific) as well as beta actin monoclonal antibody as a loading control (ThermoFisher Scientific) for overnight. Afterward, blotting was followed by incubation with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (ThermoFisher Scientific, 1/5000 dilution). The bands were detected using enhanced chemiluminescence (Abcam, USA) with the iBright™ 1500 Imaging System (Invitrogen)^{52 (link)}.