Chemi lumi one l solution

Human fibroblasts at day 10 and 27 (PDL of 10–11 and 21–22) were used for these experiments (S1A Fig). Cells were washed twice with PBS and lysed in lysis buffer containing 50 mM Tris HCl pH 7.5, 30 mM KCl, 5 mM EDTA, 1% NP-40, 1 mM dithiothreitol, and 0.1% sodium dodecyl sulfate (SDS), protease inhibitor (Nacalai Tesque, Kyoto, Japan), and phosphatase inhibitor (Pierce Thermo Fisher Scientific, IL, USA). Twenty μg of protein per lane was subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and then transferred to a polyvinylidene fluoride membrane. The membrane was incubated with primary antibody overnight at 4°C. Antibodies for total p53 and phosphorylated p53 (Ser 15) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p21 antibody and anti-β-actin antibody were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and Sigma-Aldrich (St Louis, MO, USA), respectively. After incubation with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature, signals were visualized with ImmunoStar LD (WAKO, Tokyo, Japan) or Chemi-Lumi One L solution (Nacalai Tesque, Kyoto, Japan).

Cell culture was the same as previously described. Whole-cell lysates were prepared for western blotting as we previously performed [22 ]. The samples (2 μg for ABCB1 measurement, 30 μg for others) were equally subjected to 4–15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, California, USA) and transferred to Immun-Blot PVDF Membrane (Bio-Rad). After blocking with Blocking One buffer (Nacalai Tesque, Inc., Kyoto, Japan) for 2 h, the membranes were incubated with the indicated antibodies for overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. The bands were further dealt with Chemi-Lumi One L solution (NACALAI) and analyzed. The primary antibodies for caspase-8 were obtained from Medical & Biological Laboratories CO., LTD. (Nagoya, Japan) and those for caspase-3, caspase-9, survivin, NF-kappa B p65, p-NF-kappa B p65, Bcl-xL, and Bcl-2-associated X protein (Bax) were from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). Primary antibodies for ABCB1 and GAPDH and secondary antibodies goat anti-rabbit IgG H&L (HRP) and rabbit anti-mouse IgG H&L (HRP) were purchased from Abcam (Cambridge, Massachusetts, USA). The density of bands was measured using a LAS4000 fluorescence image analysis system (FUJIFILM, Tokyo, Japan). All experiments were performed three times.