Ecl assay

Lysate proteins from adherent monocytes were subjected at 20 µg/lane to SDS-PAGE/WB. Alternatively, purified CYP4F11 was modified in vitro by 4-HNE (see below) and run alongside with non-modified CYP4F11 in each of two gels: one gel was stained immediately after SDS-PAGE by Pro Blue Safe stain (Giotto Biotech, Sesto Fiorentino, Italy) and the proteins from another gel were transferred to PVDF membrane by WB. The equality of loaded and transferred protein amounts and of protein pattern of all sample lanes was verified by Ponceau S staining. 4-HNE-protein conjugates were detected by anti-4-HNE-conjugate primary antibody (HNEJ-2), HRP-conjugated secondary antibody and ECL assay (Bio-Rad, Hercules, CA, USA). The arbitrary optical density of labelled bands was acquired by ImageLab 4.1 (Bio-Rad) for at least 4 different donors.

Cells were harvested and lysed in radioimmune precipitation assay (RIPA) buffer, 0.5M EDTA and proteases and phosphatase inhibitors cocktail total protein quantified by BCA protein assay kit (Pierce). For Western blots, 30 mg of protein extract/lane were electrophoresed, transferred to nitrocellulose membrane (Invitrogen) and incubated overnight with each of the following primary mouse monoclonal antibody: RASAL2 (1:2000; sc-390605; Lot# 2516); NF-kB (1:500; sc-8008; Lot# H1220); N-RAS (1:1000; sc-31; Lot# JQ520); TNFα (1:500; sc-515766 Lot# 17020); C-myc (1:500; sc-40; Lot# J0220); AR (1:1000; sc-7305; Lot# J2920); PTEN (1:1000; sc-7974; Lot# 10420). The GAPDH antibody (1:5000; sc-32233; Lot# J2020) was used as an internal loading control. Membranes were washed and incubated with anti-mouse secondary antibody (1:2500; sc-2005). All the antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The antigen-antibody reaction was visualized using an enhanced Chemiluminescence (ECL) assay (Bio-Rad; Hercules, CA, USA) and image the membrane using digital imager/chemidoc MP imaging system (Bio-Rad). A densitometry program using ImageJ (https://imagej.nih.gov/ij/), was used to quantify bands in the western blot and protein expression level displayed as ratio of each protein to the GAPDH protein level. The data are a representative of triplicate experiments.