Bicinchonic protein assay kit

nm‐cFib were analysed 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) and total protein content determined with the Bicinchonic Protein assay kit (Merck Group, Vimodrone, Italy); 20 μg protein were resolved on SDS‐polyacrylamide gels and electro‐transferred to a PVDF membrane (Merck Group). To quantify shed TLR4 (sTLR4), 100 μL of conditioned media were concentrated to a final volume of 25 μL and run on SDS‐polyacrilamide gels. Blots were hybridized with primary antibodies against Postn (rabbit polyclonal 19899‐1‐AP, Proteintech, UK), TLR4 (mouse monoclonal sc‐293072, Santa Cruz Biotechnology) and NRF2 (rabbit polyclonal, C‐20, Santa Cruz Biotechnology) and incubated with corresponding horseradish peroxidase secondary antibodies (Cell Signaling Technology). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck). Band intensities were determined using Alliance system (Uvitec, Cambridge, UK).

Cells 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 (Merck Group, Vimodrone, Italy) and 10–20 μg were resolved on SDS-polyacrylamide gels and electro-transferred to a PVDF membrane (Merck Group). Blots were probed using anti CCL2/MCP-1 polyclonal antibody (Novus Biologicals, Space Import-Export s.r.l., Milan, Italy), anti phospho-ERK1(T202/Y204)/ERK2 (T185/Y187) polyclonal antibody (R&D Systems, Space Import-Export s.r.l., Milan, Italy), anti phospho-p38 MAP Kinase (T180/Y182) polyclonal antibody (R&D Systems), anti phospho-JNK (Thr 183/Tyr 185) (Santa Cruz Biotechnology), reprobed with β-actin or ERK, p38, JNK (Santa Cruz Biotechnology) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merckgroup). Band intensities were determined using Alliance system (Uvitec, Cambridge, UK).

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 ug/L aprotinin, leupeptin, pepstatin A, 1 mM PMSF, and Na₃VO₄). Protein concentration was determined by using the Bicinchonic Protein assay kit (Merck, Milan, Italy) and 10–20 µg were resolved on SDS-polyacrylamide gels and electro-transferred to a PVDF membrane (Merck). Blots were probed using anti Myostatin polyclonal antibody (Proteintech Europe), anti-phospho-ERK1(T202/Y204)/ERK2 (T185/Y187) (R&D Systems, Space Import-Export s.r.l., Milan, Italy), p-NFkB p65 (Ser 536) (Santa Cruz Biotechnology), anti p-SMAD2 (Cell Signaling Technology, Euroclone, Milan, Italy), anti p-SMAD 3 (Cell Signaling Technology), p16^ink4a (St John’s Laboratory, D.B.A. Italia s.r.l.) and reprobed with β-actin or ERK (Santa Cruz Biotechnology) or SMAD 2,3 (Cell Signaling Technology) and incubated in horseradish peroxidase secondary antibodies (Cell Signaling Technology).Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Merck). Band intensities were determined using an Alliance system (Uvitec, Cambridge, UK).