Bicinchonic protein assay kit

The cell layers were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% (v/v) glycerol, 0.5% (v/v) NP-40, 1 mM EDTA) supplemented with Protease inhibitor cocktail (Merck Group). Protein concentration was determined by using the Bicinchonic Protein assay kit (Euroclone S.p.A.) and 20–30 µg was resolved on SDS-polyacrylamide gels and electro-transferred to Amersham™ Hybond™ PVDF membrane (Euroclone S.P.A.). Blots were incubated o.n. at 4 °C with monoclonal anti α-smooth muscle actin (α-SMA) antibody (clone1A4) (Merck group), re-probed with anti H3 Histone antibody (Santa Cruz Biotechnology, DBA Italia s.r.l),) or p44/42 MAPK (R&D System, Bio-Techne s.r.l., Milan, Italy) and then incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology, Euroclone s.p.a., Milan, Italy). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck Group) and band intensities were determined using Alliance imaging system (Uvitec, Cambridge, UK).

The cell layers were lysed in cold buffer (20 mM HEPES, 150 mM NaCl, 10% [v/v] glycerol, 0.5% [v/v] NP-40, 1 mM EDTA, 2.5 mM DTT, 10 µg/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF, and Na₃VO₄). Protein concentration was determined by using the Bicinchonic Protein assay kit (Euroclone S.p.A., Milan Italy) and 20 µg were resolved on SDS-polyacrylamide gels and electro-transferred to a PVDF membrane (Millipore Billerica, MA, U.S.A.). Blots were probed using antihuman BAX (Santa Cruz Biotechnology, Santa Cruz, CA, USA), antihuman XIAP (MBL, MA, USA), anti Nox 4 (Santa Cruz Biotechnology) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology), Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Millipore Billerica, MA, U.S.A.). Band intensities were determined using Alliance imaging system (Uvitec, Cambridge, U.K.).