Nf κb p65 antibody sampler kit

The whole-cell lysates were prepared with lysis buffer containing 150 mM NaCl, 3 mM NaHCO₃, 0.1% of Triton X-100 and a mixture of protease inhibitors (Roche Diagnostics, CA) plus 2 mM Na₄VO₄ and 5 mM NaF. The whole-cell lysates were homogenized with a Kontes’ Pellet Pestle, frozen in −80 °C for minimum 25 minutes and then separated from insoluble cell materials by centrifugation at 16,000 g in a bench-top Eppendorf centrifuge at 4 °C. Nuclear fractions were obtained as we described29 . Protein concentration was determined with the BCA system (Pierce, Rockford, IL). The Western blotting was performed as we previously described29 44 (link)60 (link). The primary antibody anti-β-catenin (E5) and anti-β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX). NF-κB signaling was analyzed with NF-κB p65 antibody sampler kit (Cell Signaling, Danvers, MA). The second antibody anti-mouse IgG-HRP was purchased from Santa Cruz Biotechnology. The antibody-associated protein bands were revealed with Western blotting luminol reagent (Santa Cruz Biotechnology, Dallas, TX).

All antibodies were used at a 1:1000 dilution in 5% nonfat milk for immunoblot. PE Mouse IgG1 (555749), PE Mouse Anti-Human CD13 (560998), PE Mouse Anti-Human CD133 (566593), and PE-CF594 Mouse Anti-Human CD326 (565399) were purchased from BD Biosciences. GASC1(sc-98678), c-Myc (sc-40), and SOX9 (sc-166505) were purchased from Santa Cruz Biotechnology. Nanog (ab109250), OCT4 (ab19857), SOX2 (ab97959), FBXO42 (ab81638), and GFP (ab290) were purchased from Abcam. Cleaved caspase-3 (9664), cleaved caspase-7 (8438), cleaved caspase-9 (7237), cleaved caspase-PARP (5625), Myc tag (71D10) (2278S), HA tag (2367), ROCK1 (4035), ROCK2 (9029), BCL2 (2872), ubiquitin (3936), Ki-67(9449), p-MLC2 (3671), GAPDH (2118), Rho-GTPase Antibody Sampler Kit (9968), Active Rho Detection Kit (8820), and NF-κB p65 Antibody Sampler Kit (4767) were purchased from Cell Signaling Technology.