Donkey anti rabbit hrp

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 proteins was loaded on a 12% NuPAGE (Novex, CA) and transferred onto PVDF membranes (Novex, CA) as described (Mahmoudian-Sani et al. 2017 (link); Rafiee et al. 2019 ). Membranes were blocked with 5% skim milk in Tris-buffered saline and 0.01% tween 20 (TBST buffer) for 30 min at room temperature and then incubated overnight at 4 °C with either rabbit polyclonal anti-Cyp1A1 (Abcam, CA) or mouse monoclonal anti-GAPDH (Abcam, CA), diluted in TBST buffer containing 1% skim milk. After washing in TBST, the blots were incubated with secondary donkey anti-rabbit-HRP (Abcam, CA) or goat anti-mouse-HRP (Santa Cruz, CA) antibodies for 2 h, followed by development with Pierce™ ECL Plus western blotting substrate (Thermo Fisher Scientific, USA). The bands were visualized by imaging using a LI-COR Scanner and analyzed via densitometry (LI_COR Biosciences, NE).