2.6. Pull down assay

Cells were co-transfected with Myc-HIF1α C-ter and different GST-PARP-1 domains. After following the jetPRIME protocol they were harvested in 20 nM Tris-HCl (pH 7.5), 400 mM NaCl, 20% glycerol, 5 mM DTT, 0,5 mM pefabloc and protease inhibitors (Complete Mini; Roche, Mannheim, Germany). Lysates were cleared by centrifugation and incubated for 2 h with glutathione-sepharose 4B (Sigma, St Louis, MO, USA) Beads were washed three times with 20 nM Tris–HCl (pH 7.5), 150–500 nm NaCl, 0.1% NP-40 and protease inhibitors. All samples were resuspended and boiled for 5 min in modified Laemli charge buffer (250 mM Tris-HCl (pH 7.5), glycerol 20%, SDS 10%, 1,4 M mercaptoethanol and 1% blue bromophenol). Then samples were analyzed by western blot. Blots were subsequently incubated with the anti-GST antibody.