Heat shock protein 70

Lyophilised pellets were Dounce-homogenized on ice in about 250 lL of lysis buffer [20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton], supplemented with a protease inhibitor cocktail (Roche) and phoshatase inhibitor cocktail (SIGMA). Total cell lysate (30 lg) per each cell extract was run on a 4-15% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Amersham UK). After blocking, replicate membranes were incubated overnight at 4 C with either one of the following primary antibodies: (i) Heat Shock Protein 70 (SIGMA, Cat N. H-5147) 1:1000; (ii) Heat Shock Protein 60 (SIGMA, Cat N. H-3524) 1:250; (iii) Phospho-p38 MAP Kinase (Thr180/Tyr182) (Cell Signalling, 9211) 1:250; (iv) Manganese superoxide dismutase (Enzo Life Sciences, ADI-SOD-110) 1:500. Membranes were washed three times with PBS-Tween 20 prior to incubation with a fluorescein-labelled secondary antimouse and/or -rabbit antibody (LI-COR Biosciences). Protein levels (Hsp70, Hsp60, MnSOD, P-p38 Mapk) were normalised using a-tubulin as internal control. Results were reported in arbitrary units ± SE obtained from the volumetric analysis of the normalised bands (sum of the two independent experiments).