Molecular imager gs 800 densitometer

Triplicate MEC1 protein samples (15 μg) from each condition were separated by 10% SDS-PAGE, and transferred to a PVDF membrane (Immun-Blot™, Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 5% skim milk in TBST (25 mM Tris/HCl, 137 mM NaCl, 2.7 mM KCl and 0.1% (v/v) Tween-20, pH 7.6), the membranes were incubated in TBST (4°C, 16 h) with rabbit monoclonal antibodies against BRCA1, NCL, NFKB2 p100/p52 or MYC (Cell Signalling Technology, Danvers, USA) or mouse monoclonal antibody against CCND1 (Cell Signalling Technology) and actin (Abcam, Cambridge, USA), followed by incubation (1 h, room temperature) with horse radish peroxidase (HRP)-conjugated secondary antibodies: goat-anti-mouse-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or donkey-anti-rabbit-HRP (Abcam, Cambridge, UK). Proteins were visualized using Rapid Step ECL Reagent (Merck, Kilsyth, Victoria, Australia) and ECL chemiluminescence film (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Films were scanned on a Molecular Imager GS-800™ densitometer (Bio-Rad, Hercules, CA, USA). Bands were quantified using ImageQuantTL density analysis software (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and two-tailed homoscedastic student t-tests were performed on log₂-transformed, actin-normalized ratios to the control samples.

Cell lysates were collected in RIPA buffer (50 mm Tris-HCl pH 7.5, 300 mm NaCl, 1% deoxcholate, 2% Triton-X100, 0.1% SDS). Proteins were separated by SDS-PAGE on 8% acrylamide gels prior to transfer onto a protran nitrocellulose transfer membrane. Separation of pro- and mature VWF was performed on precast Novex® (Life Technologies, Paisley, UK) 6% Tris-Glycine 1.5 mm gels. Blots were probed with primary and HRP-conjugated secondary antibodies (above). Band quantification was performed using a Molecular Imager GS-800 densitometer and Quantity One Software (version 4.6.3, BioRad, Hertfordshire, UK), normalizing against a β-actin loading control.