Enhanced chemiluminescence ecl assay kit

MCF-7 and MDA-MB-231 cells were obtained from ATCC (Manassas, VA). DMEM/F12, fetal bovine serum (FBS), Lipofectamine TM Reagent was purchased from Invitrogen (Paisley, UK). The anti pAkt, Akt, pmTOR, mTOR, GAPDH and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). The anti Beclin 1, P62, ILC3, Caspase 3, Bcl2 and Bax were purchased from Proteintech (Wuhan, Hubei, China). Enhanced chemiluminescence (ECL) assay kit was purchased from Amersham (Louisville, Co).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), TRIzol and Lipofectamine™ 2000 reagents were purchased from Invitrogen (Camarillo, CA, USA). Rg3 was provided by Dalian Fusheng Pharmaceutical Co., Ltd. (Dalian, China). It was diluted with cell culture media to final concentration. Cell Counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Annexin V/FITC kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, Jiangsu, China). Antibodies specific for PARP, caspase-3, -8, -9, Bcl-2, survivin, Bax, FUT4, β-actin, HRP-conjugated anti-rabbit and anti-mouse antibodies were purchased from Proteintech Group (Wuhan, China). Antibodies specific for p-p65, p65, pIκBα, IκBα, pIKKα/β and IKKα/β were purchased from Cell Signaling Technology (Boston, MA, USA). p65 siRNA was purchased from the Shanghai GenePharma Co. (Shanghai, China). NF-κB inhibitor (Bay 11-7082) was purchased from Selleck Chemicals (Houston, TX, USA). Nuclear extract kit and EMSA kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ChIP kit was purchased from Millipore (Billerica, MA, USA). The enhanced chemiluminescence (ECL) assay kit was purchased from Amersham Biosciences (Pittsburgh, PA, USA).

The 20 (R)-Rg3 was provided by Dalian Fu Sheng Pharmaceutical Co. (Dalian, China). A solution of Rg3 as freshly prepared in DMEM (500 μg/ml) and filtered by 0.22-μm membranes. It was diluted with cell culture media to final concentration in different treatments. DMEM, fetal bovine serum (FBS), TRIzol and Lipofectamine™ 2000 reagents were purchased from Invitrogen (Camarillo, CA, USA). DMSO and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). The enhanced chemiluminescence (ECL) assay kit was purchased from Amersham (Pittsburgh, PA, USA). Anti-rabbit FUT4, PCNA, p-ERK1/2, ERK, β-actin, HRP-conjugated anti-mouse IgM, HRP-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Proteintech group (Wuhan, China). EGFR and p-EGFR were purchased from Cell Signaling Technology (Boston, MA, USA). FITC conjugated goat anti-mouse IgG and TRITC conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-Ulex Europaeus (UEA) lectin was purchased by EY Laboratories (San Mateo, CA, USA), which preferentially recognizes the total fucose. Mouse anti-Giantin Golgi marker antibody and mouse anti-LeY antibody (BG-8) were purchased from Abcam (Cambridge, UK). AG1478 inhibitor was obtained from Sigma (St. Louis, MO, USA). Coomassie protein assay reagent was purchased from Bio-Rad (Hercules, CA, USA).