Goat anti mouse igg horseradish peroxidase conjugated secondary antibody

Proteins were detected using the following primary antibodies: anti-enterovirus VP1 clone 5-D8/1 antibody purchased from Dako (Denmark); Mouse anti-Ub, Mouse anti-MAT1, Mouse anti-CDK7, and Mouse anti-Cyclin H antibodies purchased from Santa Cruz (USA); Mouse monoclonal to RNA polymerase II CTD and Rabbit monoclonal to RNA polymerase II CTD (phosphor S5) antibodies purchased from Abcam (USA); Mouse anti β-actin purchased from Proteintech; Rabbit monoclonal CDK2 and CDK4, Rabbit monoclonal phosphor-CDK2 and CDK4, Mouse monoclonal pRb, Rabbit monoclonal pRb-phospho Ser780, pRb-phospho Ser795 and pRb-phospho Ser807/811 antibodies purchased from Cell Signaling (USA); Rabbit polyclonal Lamin B1, Mouse monoclonal c-Myc, Mouse monoclonal HA-Tag and Mouse Monoclonal Flag antibodies purchased from Sigma (USA); Goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody and goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody purchased from Thermo Fisher Scientific (USA).

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)}.

Immunoblotting was carried out as described previously [76 (link)]. The following antibodies were used: mouse anti-Flag M2 monoclonal antibody (1:1500 dilution; Sigma #F3165), rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200, Santa Cruz, CA, USA) and rabbit anti-β-Tubulin polyclonal antibody (1:2500 dilution; Abcam #ab6046, Cambridge, UK). Depending on the species of the primary antibody, the secondary antibody used was either horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:10,000 dilution; Thermo Scientific #31430) or horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution; Cell Signaling Technology #7074, Danvers, MA, USA). Blots were treated with Immobilon chemiluminescent reagent (EMD Millipore, Burlington, MA, USA) and proteins were visualized either on autoradiography film or with ChemiDoc XRS+ detection system (BioRad). Uncropped western blot images are presented in Figures S2 and S3.