Rabbit polyclonal anti p53

Mouse monoclonal anti-β-actin, rabbit polyclonal anti-bcl-2, rabbit polyclonal anti-Puma, rabbit polyclonal anti-cleaved-Bid, rabbit polyclonal anti-Bak, rabbit polyclonal anti-cleaved PARP, rabbit polyclonal anti-p53 and rabbit polyclonal anti-P21 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase linked secondary sheep anti-mouse and rabbit anti-horse IgG were obtained from GE Healthcare (Chicago, IL, USA). Propidium iodide (PI) and Annexin V were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Complete lysis-M was purchased from Roche Applied Science (Indianapolis, IN, USA). AO/EB nuclear dyes were purchased from Beyotime (Shanghai, China). Curcumin, niacin, 3-[4–dimethylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT) and other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Curcumin nicotinate (CN) was in-home synthesized. For experimental uses, CN was dissolved in DMSO at 10 mM as stock solution and then diluted to concentrations indicated.

Antibodies were as follows: mouse monoclonal anti-β-actin (AC-15), anti-β-tubulin (clone B-5-1-2) and anti-vinculin (V9131) (Sigma-Aldrich), mouse monoclonal anti-mDia1 and anti-GM130 (BD Transduction Laboratories), rabbit polyclonal anti-p300 (C-20), mouse monoclonal anti-p53 (epitope 11-25; DO-1) (Santa Cruz Biotechnology), rabbit polyclonal anti-p53 (epitope 50-100; ab17990), mouse monoclonal anti-β-catenin (#2698) (Cell Signaling Technology), rabbit polyclonal anti-mDia3 (A300-079A) (Bethyl Laboratories), rabbit polyclonal anti-Giantin (ab24586). Rabbit polyclonal anti-TGN46 (Novus Biologicals). Anti-mDia2 sera were generated in house76 .

The cytoplasmic, membrane and organelle and cytoskeletal and nuclear fractions of cells were obtained by lysing using the Cell Fractionation Kit (#9038, Cell Signaling Technology). Whole‐cell proteins were extracted by lysing in RIPA buffer. The protein lysate was separated using 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and electro‐transferred onto a polyvinylidene fluoride membrane. The membranes were blocked in 5% BSA and were incubated with primary antibodies overnight at 4°C. The following antibodies were used: mouse monoclonal anti‐NUMB (1:1000, ab14140; Abcam), mouse monoclonal anti‐MDM2 [2A10] (1:50, ab16895; Abcam, Cambridge, MA, USA), Rabbit Polyclonal anti‐P53 (1:1000, #9282; Cell Signaling Technology), Rabbit Monoclonal anti‐Histone H3 (D1H2) (1:2000, #4499; Cell Signaling Technology), Rabbit Polyclonal anti‐Caveolin‐1 (1:1000, bs‐1453R; Bioss) and mouse anti‐GAPDH (1:1000, 60 004‐1‐Ig; Proteintech Group). The membranes were washed and incubated with anti‐rabbit or anti‐mouse secondary antibody (1:5000, SSA004/SSA007; Sino Biological Inc) for 2 hours at room temperature. Finally, antigen‐antibody complexes were detected using an electrochemiluminescence Western blotting detection reagent.