Anti mcl1

Anti‐H4, anti‐ac‐Tubulin, anti‐CDK1, anti‐p‐CDK1(Y15), anti‐CDK2, ‐p‐CDK2(Y15), anti‐RRM1, anti‐RRM2, and anti‐AKT (Abcam), anti‐p‐S6(S240/244), anti‐p‐CDC25C, anti‐Wee1, anti‐c‐Myc (Cell Signaling Technologies), anti‐β‐actin, anti‐PARP, anti‐Bcl‐2, anti‐Bax, anti‐Mcl‐1, anti‐ERK (Proteintech), anti‐p‐AKT (T308), anti‐p‐AKT (S473) (Affinity Biosciences), anti‐Bim, anti‐Bcl‐xL, anti‐Bak, anti‐p‐ERK(T202/Y204), anti‐CHK1 (Selleck Chemicals), anti‐ac‐H4 and anti‐γ‐H2AX (Millipore) antibodies were used for Western blot analyses.
Western blotting was performed as described previously.³³ Briefly, whole cell lysates were prepared by sonication in 10 mmol/L Tris‐Cl, pH 7.0, containing 1% SDS, protease inhibitors and phosphatase inhibitors (Roche Diagnostics). The samples were separated by electrophoresis on SDS‐polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific). After blocking in TBS buffer (150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4) containing 5% fat‐free milk for 1 hour at room temperature, the blots were incubated with a primary antibody overnight at 4°C and then incubated with a fluorescent‐labelled secondary antibody for 1 hour at room temperature. Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li‐Cor). Western blots were repeated at least three times and one representative blot is shown.

Whole-cell lysates were extracted from cell lines treated with the indicated compounds using RIPA buffer containing phenylmethylsulfonylfluoride, a protease inhibitor, sodium orthovanadate (Santa Cruz Biotechnology), and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad). Equal amounts of whole-cell lysate were denatured (5 minutes, 95°C) in NuPAGE LDS sample buffer (Invitrogen) and separated on NuPAGE 3%–8% Tris-acetate gels or 4%–12% Bis-Tris gels (Invitrogen) prior to transfer to nitrocellulose membranes (LI-COR Biotechnology). Immunoblotted bands were detected using the ECL detection reagent (GE Healthcare). The following primary antibodies were used: rabbit polyclonal anti-BCL-2, anti-MCL-1, anti-BCL-xL, anti-BIM, anti-VAMP7, anti-RNH1, anti-p62 (Proteintech), anti-tubulin (Cell Signaling Technology), and anti-LC3B (Abcam).