Anti fth

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).