Nf kb2 p100 p52

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.

A total of 25 µg of protein was fractionated on 4–15% gradient SDS-PAGE gels and transferred to nitrocellulose membranes using the standard protocols. The following primary antibodies were used: p-ERK1/2 (#4370), p-p38 (#4511), p-JNK (#4668), p-IKB (#2859), NF-kB2 p100/p52 (#4882), Fn14 (#4403), p-AktS473 (#9271), FASN (#3180S) and CPT-1A (#12252) and were purchased from Cell Signaling Technology (Danvers, MA, USA), FABP4 (SC18661) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and anti-ß-actin was purchased from Sigma-Aldrich. The antibodies were used at the dilutions recommended by the manufacturers.