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2 protocols using primary antibodies against erk1 2 and phospho erk1 2

1

Nesfatin-1 Induced Apoptosis Pathway

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Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis. MO, USA). MES23.5 cells were provided by Professor Wei-Dong Le (Baylor College of Medicine, TX, USA). Nesfatin-1 was obtained from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA). Dulbecco’s modified Eagle’s medium nutrient mixture-F12 (DMEM/F12) was purchased from Gibco (Grand Island, NY, USA). The PE-conjugated monoclonal active-caspase-3 antibody apoptosis kit was from BD Bioscience (San Diego, CA, USA). PD98059 and the BCA kit was from Beyotime (Shanghai, CN). KT5720 and anti-rabbit secondary antibody conjugated to horseradish peroxidase were from Santa Cruz (Santa Cruz, CA). The cell fractionation kit was from Clontech (Mountain View, CA, USA). Anti-Cox IV monoclonal antibody was from Clontech (CA, USA). The primary antibody against TH was from Millipore (Darmstadt, Germany). Primary antibodies against ERK1/2 and phospho-ERK1/2 were from cell signaling technology (MA, USA). Alexa Fluor 555 donkey anti-rabbit secondary antibody was from Eugene (OR, USA). GW5074 was from Selleck (TX, USA).
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2

Regulation of Tongue SCC Progression

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Human tongue SCC Tca8113 cells were obtained from the Cell Resource Center of Peking Union Medical College (Beijing, People’s Republic of China [PRC]). Fetal bovine serum was purchased from Biological Industries (Cromwell, CT, USA). An miRNA-isolation kit, miRNA RT kit, and miRNA qRT-PCR kit were all purchased from HaiGene Inc (Harbin, PRC). A cell-cycle assay kit and annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit for apoptosis analysis were also purchased from HaiGene. The primary antibodies against RASA1, SPRED1, β-actin, and secondary antibodies were all purchased from Santa Cruz Biotechnology Inc (Dallas, TX, USA), and primary antibodies against ERK1/2 and phospho-ERK1/2 were purchased form Cell Signaling Technology (Danvers, MA, USA). The transfection reagent Lipofectamine 2000 was obtained from Thermo Fisher Scientific (Waltham, MA, USA), and miR182 mimics, anti-miR182 oligonucleotides, and corresponding controls were all obtained from Ruibo Inc (Guangzhou, PRC). CCK-8 reagent was purchased from Dojindo (Kumamoto, Japan). Matrigel basement membrane matrix was purchased from BD (Franklin Lakes, NJ, USA). An active Ras pull-down assay kit was purchased from EMD Millipore (Billerica, MA, USA). A luciferase-activity assay kit was obtained from Promega Corporation (Fitchburg, WI, USA).
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