Nxa931

In human tissue culture cells, after 72 hours of siRNA knockdown, protein was harvested by scraping, rinsed in PBS, and lysed by vortexing in AZ lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1% Igepal, 0.1% SDS, 5 mM EDTA pH 8.0) containing protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche, 11697498001) for 15 minutes at 4°C. The lysate was spun at 21,000 x g for 15 minutes at 4°C. The supernatant was removed, and total protein was quantified by Bradford assay (Bio-Rad). Samples were analyzed via SDS-PAGE. Antibodies used include α-p53 (1:5,000 Santa Cruz, sc-126 HRP), α-β-actin (1: 30,000 Sigma-Aldrich A1978), α-PAX9 (1:1,000, Cell Signaling Technology D9F1N), α-vinculin (1:20,000 Abcam ab18058), and α-puromycin (1:10,000 Kerafast 3RH11). HRP conjugated secondary antibodies include α-mouse (1:10,000 GE Healthcare NXA931) and α-rat (1:10,000 GE Healthcare NA935V). Images acquired either by film developing or by digital imaging using the BioRad ChemiDoc Imaging System.
For X. tropicalis western blotting, protein was harvested according to the TRIzol (Life Technologies 5596018) protocol for 2 replicates, and as in [70 (link)] for 1 replicate. Samples were analyzed via SDS-PAGE. Antibodies included α-p53 (1:800, Thermo Fisher MA1-12549) and α-GAPDH (1:5,000 Ambion AM4300). HRP conjugated secondary antibodies include α-mouse (1:10,000 GE Healthcare NXA931).

Cells were homogenized, and extracts were prepared using lysis buffer (NER buffer from the NER-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), 1× protease inhibitor cocktail, 1 μM microcystin-LR). The soluble fraction was recovered by centrifugation at 16,000 × g for 10 min at 4 °C. Protein concentration was measured with the BCA Protein Assay Kit (Pierce Biotechnology), and 30 μg of protein from each sample was resolved by SDS-PAGE. The resolved bands were transferred onto a nitrocellulose membrane and subjected to western blotting with the appropriate antibodies.
The following primary antibodies were used: mouse anti-CK1δ (C-8) (1/1000, Santa Cruz Biotechnology, sc-55553), goat anti-CK1δ (N-19) (1/500, sc-6475, Santa Cruz Biotechnology), rabbit-anti C-terminal Brd4 (1/2000)^{19 (link)}, rabbit anti-Brd4 (1/1000, A301-985A50, Bethyl Laboratories Inc.), rabbit anti-phospho-Brd4 492/494 (1/2000, ABE1453, Millipore), mouse anti-Gapdh (1/5000, NB300-221, Novus Biolechne), goat anti-CK2α (1/500, sc-6479, Santa Cruz Biotechnology), mouse anti-Flag (1/5000, A8592, Sigma), and rabbit anti-cyclin D1 (1/1000, ab134175, Abcam). The following secondary antibodies were used: anti-goat IgG–HRP (1/1000, 7074, Cell Signaling), anti-mouse IgG–HRP (1/1000, NXA931, GE Healthcare) and anti-rabbit IgG–HRP (1/1000, NA9340V, GE Healthcare).