Protein Extraction and Western Blot Analysis

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).