Anti rabbit igg and hrp linked antibody

Calyxin Y was isolated from A. katsumadai as described before [14 (link)]; CDDP was purchased from Sigma-Aldrich (St. Louis, MO, USA), and each had a purity of > 99%. Both compounds were dissolved in DMSO at a stock concentration of 50 mM, and were stored at -20 °C. Cells were treated with DMSO as a control. MDC and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 7-AAD was purchased from Yeasen Biotechnology (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum were obtained from Thermo Fisher Scientific (Fair Lawn, NJ, USA). Primary antibodies against eEF2k, eEF2, phospho-eEF2 (Thr56), β-TrCP (D13F10), cleaved caspase-3 (Asp175), caspase-3, cleaved caspase-7 (Asp198), caspase-7, cleaved PARP (Asp214), PARP, Bcl-xL ((54H6), Bax (D2E11), AIF (D39D2), cytochrome c (6H2.B4), p62 (D5E2), β-Actin (13E5); and anti-rabbit IgG and HRP-linked antibodies and anti-mouse IgG and HRP-linked antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The TG2 antibody was purchased from Abcam (Cambridge, MA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA).

The right kidneys were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor cocktail and centrifuged at 12,000 rpm for 10 min at 4 °C. The protein content in the supernatants was quantified using the BCA protein assay (TaKaRa BIO INC, Tokyo, Japan). After pretreatment by heating with sample buffer, samples containing 20 µg of protein were loaded onto 15% sodium dodecyl sulfate polyacrylamide gels, followed by electrophoretic transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by treatment with 5% skim milk for 60 min at room temperature. After washing the membranes three times with PBS containing 0.05% Tween-20, the membranes were incubated with rabbit anti-caspase 3 primary antibody (cat#: c8487; Sigma-Aldrich, St. Louis, MO, USA), or β-actin antibody (cat#: 4967; Cell Signaling Technology, Beverly, MA, USA) for 60 min at room temperature. The membranes were then washed with PBS containing 0.05% Tween-20, and treated with anti-rabbit IgG and HRP-linked antibodies (cat#: 7074; Cell Signaling Technology, Beverly, MA, USA) for 60 min at room temperature. All band images were acquired using the LAS 4000 system (Fujifilm, Tokyo, Japan). All band intensities derived from cleaved caspase-3 and β-actin were analyzed using ImageJ.