G box chemi gel imaging systems

Cytosolic and nuclear protein fractions (60 μg) were electrophoresed on 3–8% Tris-acetate precast gels (Invitrogen, Life technologies, Carlsbad, California, USA) as previously described [24 (link)]. Blots were incubated with rabbit anti human-GR total (1:1500) (Bethyl Laboratories, Montgomery, TX, USA, S1 Table) antibody targeted to residue 150–200 of the GR receptor and expected to cross react with other animal species including bovine, ovine, equine and primate. The appropriate secondary antibody (goat anti-rabbit, goat anti-mouse or donkey anti-goat 1:2500) was applied for 1 hr. Membranes were subsequently probed with anti-β actin (1:2500, Abcam laboratories, UK, S1 Table) and anti-lamin A/C (1:1500, Santa Cruz Biotechnology, Santa Cruz, California, USA, S1 Table) antibodies as loading controls for cytoplasmic and nuclear fractions, respectively. The densitometric analysis was carried out using G:BOX Chemi Gel Imaging Systems (SYNGENE) to quantify the expression levels of different GR isoforms relative to β actin. Peptide competition with anti GR total antibody (1μg/1.5ml) incubated with 1X (1μg) and 2X (2μg) concentration of the control peptide (Bethyl Laboratories, USA) was performed as a specificity control.