Enhanced chemiluminescence ecl system

RCC cells were incubated 40 h in serum-free medium and then exposed to sCD40L (100 ng/mL) and sCD40L enhancer (1 μg/mL) or the enhancer alone for the indicated time periods. At the end of the treatment, the cell monolayer was lysed in RIPA buffer as previously reported [38 (link)]. Aliquots containing 40 μg of proteins from each lysate were subjected to SDS/PAGE on a 7.5% or 10% gel, according to the molecular weight of the protein of interest, under reducing conditions and then electro-transferred onto nitrocellulose membrane (Hybond^TM C, Amersham, Cologno Monzese, Italy). The membrane was blocked over-night at 4 °C with PBS plus 0.1% Tween-20 and 5% low-fat milk and then incubated with the relevant primary antibodies at the appropriate concentration. The membranes were then incubated for 1 h at RT with peroxidase-conjugated secondary antibodies. The membranes were washed three times at RT in TBS and then once with 0.1% SDS in PBS. The ECL enhanced chemiluminescence system (Amersham) was used for detection. The same membranes were then stripped and immunoblotted again with appropriate antibodies to normalize the protein quantity. The ECL enhanced chemiluminescence system (Amersham) was used for detection. Intensity of the bands was quantified using ImageJ software. Phosphorylated protein expression was normalized to total protein.

Total cellular extracts and soluble nuclear extracts from HRPTECs were prepared as described previously^{57 (link),58 (link)}. Western blotting was carried out using 3–8% Novex NuPAGE Tris-acetate gels (Invitrogen) for HIF-1α and nucleoporin p62 or 4–12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen) for p-AMPKα (Th172), AMPK and α-actinin under reducing conditions. After proteins were transferred onto a Hybond-P PVDF membrane (Amersham Biosciences Co., Piscataway, NJ, USA), the membranes were incubated with the primary antibodies (dilution 1:1000), incubated with a peroxidase-conjugated secondary antibody (dilution 1:50000) (Amersham), and visualized with an enhanced chemiluminescence (ECL) system (Amersham). Selected blots were washed and reprobed with an antibody against nucleoporin p62 for nuclear protein extracts and α-actinin for total cellular extracts to control for small variations in protein loading and transfer. Images were processed using ImageJ (U. S. National Institutes of Health, Bethesda, MD, USA) for densitometric analysis. Signal intensities in the control lanes were arbitrarily assigned a value of 1.00.

Flower of Pueraria lobata Ohwi was purchased from Inno Hanbang Herbal Drug Co. Ltd. (Seoul, Korea) and a voucher specimen of the herb (PF1-2012) was deposited in the herbarium of the College of Pharmacy, Kyung Hee University. Pueraria flower (100 g) was added to 10 times of distilled water, heat-extracted (for 2 h at 100 °C), and concentrated under reduced pressure. The filtrate was lyophilised using an FDU-550R freeze-dryer (Eyela Co., Tokyo, Japan), and stored at 4 °C until use. The dried extract of Pueraria flower (PFE) was dissolved in distilled water before each experiment. Foetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, penicillin G, and streptomycin were obtained from Life Technologies (Grand Island, NY, USA). CellTracker™ was purchased from Invitrogen (Grand Island, NY, USA). RNase, hydrocortisone, and PD98059 were obtained from Sigma Chemical Co. (St. Louis, MO, USA.). MMP-2 antibody was from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies for β-actin, MMP-9, total extracellular signal-regulated kinase (t-ERK), phospho-ERK (p-ERK), and the peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Enhanced chemiluminescence (ECL) system was purchased from Amersham Pharmacia Biotech (Oakville, ON, Canada).