Rabbit anti β tubulin h 235

Following relevant treatment of cells, protein was either harvested on ice in RIPA buffer containing a fresh mixture of complete protease inhibitors (Roche, Switzerland) and 0.1 M Sodium Orthovanadate to inhibit phosphatase activity or fractionated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer's instructions (Thermo Scientific, USA). Protein concentrations were determined using the Bicinchoninic Acid (BCA) assay kit (Pierce, Thermo Scientific, USA). Western blot analysis was performed using rabbit anti-KPNB1 (H-300) (sc-11367, Santa Cruz Biotechnology, USA), rabbit anti-NFĸB p65 (H-286) (sc-7151x, Santa Cruz Biotechnology, USA), rabbit anti-NFĸB p50 (H-119) (sc-7178x, Santa Cruz Biotechnology, USA), rabbit anti-p-c-Jun (Ser63/73) (sc-16312-R, Santa Cruz Biotechnology, USA), rabbit anti-c-Jun (D) (sc-44, Santa Cruz Biotechnology, USA), mouse anti-GAPDH (0411) (sc-47724, Santa Cruz Biotechnology, USA), rabbit anti-β-tubulin (H-235) (sc-9104, Santa Cruz Biotechnology, USA) and rabbit anti-TBP (N-12) (sc-204, Santa Cruz Biotechnology, USA).

For protein extraction, cells were washed with PBS and lysed using RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% (w/v) sodium deoxycholate, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 2 mM EGTA, 2 mM EDTA, 50 mM NaF, 5 mM Na₂P₂O₇, 1 X complete protease inhibitor cocktail (Roche) and 0.1 M Sodium Orthovanadate). For PARP cleavage analysis, dead cells were collected by centrifugation and combined with live cell lysates. Lysates were sonicated, centrifuged, and the supernatant quantified using the BCA Protein Assay Kit (Pierce, Thermo Scientific) according to the manufacturer’s instructions. Proteins were subjected to Western blot analysis using the following antibodies: rabbit anti-Kpnβ1 (H-300, sc-11,367, Santa Cruz), rabbit anti-β-tubulin (H-235, sc-9104, Santa Cruz), rabbit anti-PARP1/2 antibody (H-250, sc-7150, Santa Cruz), mouse anti-GAPDH (0411, sc-47,724, Santa Cruz), rabbit anti-p21 (H-164, sc-756, Santa Cruz), rabbit anti-Mcl-1 (H-260, sc-20,679, Santa Cruz), mouse anti-cyclin D1 (HD11, sc-246, Santa Cruz), rabbit anti-c-Myc (N-262, sc-764, Santa Cruz), mouse anti-p53 (DO-7, M7001, DakoCytomation), mouse anti-XIAP (610,763, BD Biosciences), and rabbit anti-phospho-Histone H2AX (γH2AX, Ser139, 20E3, #9718, Cell Signal).