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. Non-specific reactivity was blocked in non-fat dry milk in Tween-PBS (5% (w/v) milk in phosphate-buffer saline (PBS) (pH 7.4) and 0.005% Tween 20) for 2 h at room temperature. The nitrocellulose membranes were incubated overnight at 4 °C with the following antibodies: (a) anti-FHC (H-53) (1:200; sc-25617, Santa Cruz Biotechnology, Dallas, TX, USA), (b) anti-p65 (C-20) (sc-372, 1:1000; Santa Cruz Biotechnology), (c) anti-HDAC (AV38530, 1:5000; Sigma-Aldrich), (d) anti-HA probe (F-7) (sc-7392, 1:1000; Santa Cruz Biotechnology), (e) anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology), (f) anti-phospho-IκBα (Ser 32/36) (1:1000; sc-101713, Santa Cruz Biotechnology), and (g) anti-IκBα (C-21) (1:1000; sc371, 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).
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).