Enhanced chemiluminescence ecl detection reagents

The human ovarian cancer cell lines, HEY, SKOV3, OVCAR5, IGROV1 and OV433 were used. The SKOV3 and OVCAR5 cell lines were maintained in DMEM/F12 medium with 5% fetal bovine serum (FBS). The IGROV1 and OV433 cell lines were maintained in RPMI 1640 with 10% FBS. The HEY cell line was maintained in RPMI 1640 with 5% FBS. All medium was supplemented with 100 U/ml of penicillin and 100 ug/ml of streptomycin. The cells were cultured in a humidified 5% CO2 at 37°C. For glucose studies, the SKOV3 and OVCAR5 cell lines were cultured in DMEM medium without glucose (Cat # 11966–025, Gibco), containing 10% dialyzed FBS and supplied with specific concentrations of glucose (1 mM, 5 mM and 25 mM).
Glucose solution, MTT and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) was bought from Life Technologies (Grand Island, NY). Enhanced chemiluminescence (ECL) detection reagents were purchased from GE Health care (Piscataway, NJ). The non-fat milk, 20% bull serum albumin (BSA) and RNase A were purchased from Sigma (St. Louis, MO, USA), and all the primary antibodies for phosphorylated-S6, pan-S6, phosphorylated-AKT, pan-AKT, cyclin D1, CDK6, p21, Bcl-2, Mcl-1, Ki-67, VEGF, cleaved caspase 3, perk, bip, calnexin, MMP-9 and α-tubulin were obtained from Cell Signaling Technology (Danvers, MA, USA).

The cell lines used in this study were ECC-1, KLE, RL95-2, Ishikawa, AN3CA, and Hec-1a. For the ECC-1 cells, RPMI 1640 medium with 5% fetal bovine serum (FBS) was used. For the KLE and RL95-2 cells, DMEM/F12 with 10% FBS was used. For the Ishikawa and AN3CA cell s, MEM with 5% FBS was used. For the Hec-1a cells, McCoy's 5A with 10% FBS was used. All cells were purchased from ATCC (American type culture collection, USA) and cultured at 37°C in a humidified incubator containing 5% CO₂. All the reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. JQ1 was kindly gifted by Dr. James E. Brander (Harvard Medical School, MA). All the primary antibodies for phosphorylated-S6, pan-S6, phosphorylated-AKT, pan-AKT, cyclin D1, CDK6, CDK4 and Ki-67 were obtained from Cell Signaling Technology (Danvers, MA, USA). Enhanced chemiluminescence (ECL) detection reagents were purchased from GE Health care (Piscataway, NJ).

Cells were washed 3 times with PBS (4°C) and lysed in RIPA buffer (ProteinSimple). Cell lysates were heated to 95°C in SDS loading buffer, and the protein concentration was determined using the RCDC protein detection assay (Bio-Rad). Proteins separated by SDS-PAGE were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), blocked in 5% skim milk, and incubated with primary and secondary antibodies. p38/MAPK (1 : 1000, Cell Signaling Technology), p-p38/MAPK (1 : 1000, Thr180/182, Cell Signaling Technology), and GAPDH-HRP (1 : 5000, Proteintech) antibodies were used. Bands were visualized by chemiluminescence using the ChemiDocTM XRC⁺ Imaging System (Bio-Rad) and enhanced chemiluminescence (ECL) detection reagents (GE Healthcare).