Donkey anti rabbit igg

Dulbecco’s modified eagle’s medium (DMEM) and Fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY). PGE1, hemin, Hoechst 33342, 2’7’- dichlorofluorescein diacetate (DCFH-DA), trypsin, and In Situ Cell Death Detection Kit was purchased from Sigma (St. Louis, MO). The antibodies of Anti-NeuN, anti-HO-1, anti-Nrf2, anti-Cleaved caspase-3, anti-Bcl-2, anti-Bax, anti-Cyt C, anti-β-actin, and TMRE-Mitochondrial Membrane Potential Assay Kit were purchased from Abcam (Cambridge, MA). HRP-conjugated goat anti-mouse IgG or donkey anti-rabbit IgG were obtained from Cell Signaling Technology (Danvers, MA). The kits of Reactive Oxygen Species Assay and Caspase-3/CPP32 Colorimetric Assay were provided by Beyotime (Nantong, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan).

Disodium hydrogen phosphate (Na₂HPO₄), sodium bicarbonate (NaHCO₃) and potassium dihydrogen phosphate (KH₂PO₄) were bought from Honeywell Specialty Chemicals Seelze GmbH. FBS and Trypsin-EDTA were obtained from AG Scientific. The Annexin V-FITC apoptosis detection kit was bought from Strong Biotech Corporation. DMSO, hydrochloric acid, Tween-20, SDS, sodium hydroxide DMA, NaAsO₂, staurosporine, high-glucose DMEM, PI, RNase A, MTT and penicillin-streptomycin were purchased from MilliporeSigma. A Micro BCA protein assay kit was purchased from Thermo Fisher Scientific, Inc. HEPES, potassium chloride, sodium chloride and Tris base were procured from J.T. Baker. Antibodies against β-actin (cat. no. 58169; 1:5,000), JNK (cat. no. 9252), phosphorylated (p)-JNK (cat. no. 9251), ERK1/2 (cat. no. 9102), p-ERK1/2 (cat. no. 9101), p38 (cat. no. 9212), p-p38 (cat. no. 9215), cleaved caspase-8 (cat. no. 9429), cleaved PARP (cat. no. 9542), cleaved caspase-9 (cat. no. 9509) and cleaved caspase-3 (cat. no. 9661) were attained from Cell Signaling Technology, Inc. Donkey anti-rabbit IgG (cat. no. NEF81200-1EA) conjugated to HRP was bought from PerkinElmer, Inc. ECL detection kit was obtained from MilliporeSigma.

Protein lysates (50 µg per lane) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies (1:500 dilution) at 4°C overnight, followed by horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution). Anti-human RBM8A (Santa Cruz, sc-32312), anti-human actin (Proteintech, HRP-60008), anti-human snail (Cell Signaling Technology, 3879), anti-human p-stat3 (Cell Signaling Technology, 9131), anti-human stat3 (Cell Signaling Technology, 12640), anti-human fibronectin (Sigma-Aldrich, F3648), anti-human vimentin (Cell Signaling Technology, 5741), anti-human Notch (Abways Technology, CY52444) and donkey anti-rabbit IgG (Cell Signaling Technology, 7074) were used in this study. Immune-reactive proteins were visualized by enhanced chemiluminescence (ECL).