Anti β actin 20 33

Whole protein extracts from cells at 24 h following siRNA transfection or untransfection were lysed in NaCl, 1% Nonidet‑40, 50 mM Tris‑HCl (pH 7.5) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific), and protein concentrations were determined using a Bio-Rad protein assay system (Bio-Rad). Equivalent amounts of proteins were separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Bio-Rad). After being blocked in Tris-buffered saline (TBS) containing 5% non-fat milk, the blots were incubated with the following primary antibodies: anti-FOXM1 (clone sc-502, 1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), and anti-β-actin ((20–33), 1:200 dilution) (Sigma-Aldrich, Saint Louis, Missouri, USA), at 4 °C for 12 h, followed by incubation with horseradish peroxidase-conjugated secondary (anti-mouse or anti-rabbit) IgGs at room temperature for 1 h. Signals were detected on BioSpectrum® Imaging System (UVP, LLC, Upland, CA, USA) with the LiteAblot® EXTEND (EuroClone). Images were processed with VisionWorks® LS Image Acquisition and Analysis software (version 7.0.1, UVP, LLC).

Immunoblotting analysis was performed as described previously [19 (link)]. Whole-cell extracts (50–100 µg protein) were prepared and resolved on SDS-PAGE (XCell SureLock Mini-Cell and NuPAGE Bis-Tris Precast Gel, Thermo Fisher Scientific). Gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked, and incubated with primary antibodies for 16 h. Membranes were washed and incubated with HRP-coupled secondary antibodies. The proteins were detected via chemiluminescence using the ECL Select Western Blotting Detection Reagent (Cytiva, Tokyo, Japan). Chemiluminescence measurements were performed using an ImageQuant LAS4000 system (FUJIFILM, Tokyo, Japan). The following antibodies were used: anti-DNA-PKcs (E6U3A), anti-caspase-3, anti-DNA Ligase IV (D5N5N), anti-ATM antibody (D2M2), anti-phospho-ATM-Ser1981 (D25E5), anti-PARP, anti-phospho-p53-Ser 15, anti-p21 (12D1) antibody (Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-β-Actin (20–33) (Sigma-Aldrich; 1:4000), anti-cyclin B (BD Transduction Laboratories, Franklin Lakes NJ, USA; 1:1000), anti-tubulin (B-5-1-2) (Sigma-Aldrich; 1:4000), anti-N-Myc (B8.4B), anti-p53 (DO-1) (Santa Cruz Biotechnology; 1:500, Dallas, TX, USA), and horseradish peroxidase-coupled anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology; 1:2000).