Adi spa 803

HSP27 in the cytosolic and cytoskeletal fractions was measured as previously described in detail [5 (link)]. Briefly, HSP27 was detected using a in house-made double antibody sandwich ELISA. By using capture antibodies (25 ng/well; ADI-SPA-800, Enzo Life Sciences) and detection antibodies against HSP27 (ADI-SPA-803, Enzo Life Sciences), HSP27 was determined by using a filter photometer (Expert 96, ASYS Hitech) measuring optical density at 450 nm.

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.