Adi spp 715

Muscle HSP was assessed by Western blotting for muscle HSP25 and HSP72 content using primary polyclonal antibodies specific to muscle HSP25 (ADI-SPP-715, Enzo, Farmingdale, USA) and muscle HSP72 (ADI−SPA−812, Enzo, Farmingdale, USA), diluted 1:1000 in TBST and 2% milk, with secondary antibody diluted 1:20,000 in 3% milk. The overall Western blotting protocol has been previously described [45 (link)].

HSP70 monoclonal antibody (SPA-810), HSP27 monoclonal antibody (ADI SPA-800), human recombinant HSP70 protein (SPP-755), polyclonal HSP70 antibody (SPA-812), recombinant human HSP27 (ADI-SPP-715), and polyclonal HSP27 antibody (ADI- SPA-803), were from Enzo Lifesciences (Antwerp, Belgium). Ficoll was from Nycomed (Oslo, Norway), Bovine serum albumin (BSA) was from Roche (Boehringer Mannheim, Germany), Fetal calf serum was from Biochrom (International Medical, Wavre, Belgium), RIPA buffer was made up of 1% nonidet P40, 1% deoxycholic acid, NaCl 150 mM, 0.1% SDS, 1% Triton X-100, phosphate inhibitors (50 mM NaF, 10 mM h-glycerophosphate, 1 mM Na₂P₂O₇ 10H₂O, 10 mM Na₃VO₄, 10 mM p-nitrophenylphosphate) and a cocktail of protease inhibitors (10:1000). Fix and Perm cell permeabilization kit was from IMTEC (Antwerp, Belgium). Phosphate-buffered saline (PBS) consisted of 40 mM Na 2HPO₄ 2H₂O, 135 mM NaCl, 3 mM KCl, and 1 mM KH₂PO₄.