This study was approved by Stockholm's ethic vetting board. Postmortem samples from the orbitofrontal and dorsolateral prefrontal cortices, Brodmann's areas 47 and 9, respectively, of 15 human controls and 15 alcoholics were obtained from the New South Wales Tissue Resource Center. Cases were collected by qualified pathologists under full ethical clearance and with informed, written consent from the next of kin. All subjects were male and Caucasian. Alcoholics drank ≥ 80 g of ethanol per day in average, met DSM-IV criteria, did not have Wernicke–Korsakoff's syndrome or liver cirrhosis and had no history of poly drug abuse. Controls had no history of drug abuse. Individual demographic and clinical data for all subjects are given in supporting information Table S1
Cell culturing and transfection
Human HeLa carcinoma cells were grown in Iscove's Modified Dulbecco's Media, supplemented with 10% Fetal Bovine Serum in a 37°C incubator with 5% CO2. Mouse FosB and ΔFosB cDNAs were subcloned into the mammalian expression vectors pcDNA™3.1 / Hygro (+) and pcDNA™3.1 (Invitrogen, Carlsbad, CA), respectively, to generate the plasmids YOF235 and YOF254. Transfections were carried out using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. Mock-transfected cells were used as negative controls.
Cell and tissue extracts were prepared by solubilization of pelleted cells and powdered tissue, respectively, in SDS buffer, and sonication. DC assay (Bio-Rad, Hercules, CA) was used for determination of protein concentration. Cell and tissue homogenates were resolved by SDS-PAGE on 10% Tricine gels. Proteins were transferred onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) and stained with MemCode (Pierce, Rockford, IL). All blots were made in duplicates, probed with the following antibodies: rabbit polyclonal anti-FosB (cat. # sc-48, Santa Cruz, CA) at 1:200; mouse monoclonal anti-FOSB (cat. # ab11959, Abcam, Cambridge, UK) at 1:500; and mouse monoclonal anti-FOSB (cat. # LS-C528, Lifespan, Seattle, WA) at 1:333; and incubated with the appropriate peroxidase-conjugated secondary antibody (cat. # 170-6515 and 170-6516, Bio-Rad) at 1:25000. sc-48 was blocked by preincubation with 10−7 M of blocking peptide (cat. # sc-48 P, Santa Cruz). Blots were developed in Amersham's Enhanced Chemiluminescence System (Amersham, Little Chalfont, UK). Films were digitized using a scanner. Densitometric analysis was performed in Image Gauge (V3.12) (Fujifilm, Tokyo, Japan). To enable data normalization, reference samples consisting of pooled OFC tissue extracts from all subjects were loaded onto three wells: the second from the left and right edge, respectively, and the middle one. Protein optical densities (ODs) were expressed as ratio to MemCode™ OD.
Data normality was examined with Shapiro-Wilk's test. Depending thereon, group differences were investigated either with Student's t-test or Mann-Whitney U test. Likewise, correlations of protein immunoreactivities with age, postmortem interval (PMI), brain pH and storage time were examined either with Pearson's correlation or Spearman's rank correlation. Covariate influence was assessed with analysis of covariance (ANCOVA). Significance was set to P < 0.05. Statistical analysis was performed in Statistica (V8.0) (StatSoft, Tulsa, OK).