Ecl western blotting analysis system

TOV-21G and A2780 cells were seeded in a six-well plate (5 × 10⁵ cells per well). Following overnight incubation, ACY-241 and PCI-34051 were treated alone or in combination for 24 h. Cells were washed with ice-cold 1× PBS and lysed with 100 µL lysis buffer (0.5% NP-40, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 5 mM EGTA, 120 mM NaCl, 25 mM NaF, 25 mM glycerol phosphate, 1 mM PMSF, and 1 mM bezamidine). Bradford protein assay was used to measure the protein concentrations of cell lysates. Samples were prepared with a 5× sample buffer and subjected to SDS-PAGE on a polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane and blocked with 5% skim milk at room temperature for 1 h. The membranes were incubated with primary antibodies at 4 °C overnight. Membranes were washed three times with 0.1% Tween-20/PBS and incubated with anti-mouse or anti-rabbit secondary antibody coupled to HRP for 3 h at room temperature. Protein bands were visualized using the ECL Western blotting analysis system (Thermo Scientific Pierce, Waltham, MA, USA).

2A3 and FaDu cells were seeded at a density of 5 × 10⁵ cells and treated with 4 μM ACY-241 and 2 μM JQ1 on the next day. Cells were washed twice with ice-cold PBS and extracted with 100 μL lysis buffer, then lysed by sonication at 20% amplitude. Bradford protein assay was performed to measure protein concentrations. Protein samples were prepared with a 5× sample buffer and loaded to 7.5–12% polyacrylamide gel. After SDS-page, proteins were transferred to nitrocellulose membrane. Membranes were blocked with 5% skim milk at room temperature and incubated with primary antibody against α-tubulin (1:1000), HDAC6 (1:1000), c-Myc (1:250), p-AKT (1:500), AKT (1:1000), p65 (1:1000), TNF-α (1:250), GAPDH (1:10,000) and p-p65 (1:1000), MMP-2 (1:500), MMP-9 (1:1000), PARP (1:1000), XIAP (1:1000), caspase-3 (1:500), Bcl-xL (1:500), MT1-MMP (1:500), acetyl α-tubulin (1:2000), and Snail (1:2000) overnight at 4 °C. Membranes were washed with 0.1% Tween-20/PBS and incubated for 3 h with an anti-rabbit/mouse secondary antibody coupled to HRP. Bound antibodies were detected with the ECL Western blotting analysis system (Thermo Scientific Pierce).

A2058 cells were seeded at 5 × 10⁵ cells per well in a six-well plate. After overnight incubation, the cells were treated with 0.1% DMSO or 20 µM PCI-34051 for 24 h. A2058 HDAC8 KO and OE cells were seeded at 5 × 10⁵ cells per well in a six-well plate and incubated for 24 h. Hypoxia was induced using 100 µM CoCl₂ for 12 h or by placing the cells in a hypoxia induction chamber. The cells were washed with ice-cold 1× PBS and lysed with 100 µL lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 25 mM glycerol phosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1% NP-40, 1 µg/mL leupeptin, 1 µg/mL aprotinin, 1 mM PMSF, 1 mM benzamidine). The protein concentrations of the cell lysates were measured using a Bradford assay. Samples were prepared with a 5× sample buffer and boiled for 5 min. The prepared samples were loaded onto a polyacrylamide gel and subjected to SDS-PAGE. Then, the gel was transferred onto a nitrocellulose membrane. After blocking the membrane with 5% skim milk, it was incubated with primary antibodies at 4 °C overnight. Membranes were washed with 0.1% Tween-20/PBS and incubated with an anti-mouse or anti-rabbit secondary antibody coupled to HRP at room temperature for 3 h. The ECL Western blotting analysis system (Thermo Scientific Pierce, Waltham, MA, USA) was used for detection.