Anti γ tubulin c 20

Cell lysates having equal protein concentrations were loaded on a Criterion TGX 4–20% precast polyacrylamide gel (Bio-Rad) and transferred onto a PVDF membrane. Primary antibodies: anti-CENP-F (ab5) (Abcam), anti-BUB1 (B3), anti-ZW10 (3363C4a), anti-vinculin (H-300), anti-γ-tubulin (C-20), anti α-tubulin (Santa Cruz Biotechnology) and anti β-actin (Sigma Aldrich). HRP-conjugated secondary antibody (Cell Signaling Technology) was used. ImageJ (Fiji software, https://fiji.sc/) were used to quantify western blot signals.

Cells (cultured with or without AGI-5198) were plated at a density of 300,000 cells/well in 6-well plates and left to adhere overnight. After 24 h incubation with EGCG (0, 50, or 100 μM), cells were irradiated with 0, 2, or 4 Gy. After 30 min, cytosolic extracts were prepared in 1× RIPA buffer (Cell Signaling Technologies) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell extracts were sonicated to release nuclear proteins. Protein samples (25 μg) were electrophoresed on 10% SDS-PAGE gels and electroblotted onto nitrocellulose (GE Healthcare). Blots were stained with anti-γH2AX antibody (Ser139; #2577; Cell Signaling Technologies) and anti-γ-tubulin (C20) (Santa Cruz Biotechnology, Dallas, TX, sc-7396), followed by appropriate secondary antibodies labeled with IRDye680 or IRDye800 (ThermoFisher). Signals were visualized and quantified using the Odyssey system (Li-COR, Lincoln, NE).

A total of 40 μg protein extract was boiled for 10 min in SDS sample buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting as reported in Di Sanzo et al. (38 (link)). The nitrocellulose membranes were incubated overnight at 4°C with the following antibodies: (a) anti-CXCR4 (1:500; Abcam), (b) anti-HIF-1α (H-206) (1:200; Santa Cruz Biotechnology), (c) anti-p65 (C-20) (sc-372, 1:1,000; Santa Cruz Biotechnology), (d) anti-HDAC (1:5,000; Sigma-Aldrich), (e) anti-HA probe (F-7) (1:1,000; Santa Cruz Biotechnology), (f) anti-Vimentin, (g) anti-E-cadherin, (h) anti-Snail, (i) anti-Slug (1:1,000; Cell Signaling Technology, Danvers, MA, USA), (l) anti-FtH (1:200; Santa Cruz Biotechnology), (m) anti-γ-Tubulin (C-20) (1:2,000; Santa Cruz Biotechnology), (n) anti-Nucleolin (D4C7O) (1:1,000; Cell Signaling Technology) over-night at 4°C, followed by incubation with goat anti-rabbit and mouse anti-goat secondary antibodies (1:5,000; Santa Cruz Biotechnology). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactive bands were visualized with the ECL Western blotting detection system (BioRad, Hercules, CA, USA).