P nfkbp65 ser536

Total protein was extracted and quantified from heart tissue using the radioimmunoprecipitation assay (RIPA) buffer containing 0.5% protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, #P8340 #P0044). Proteins were denatured and separated on polyacrylamide gels (BioRad, #4561093EDU) using SDS‐PAGE before transfer on PVDF membranes (ThermoScientific, Waltham, MA, #PI88585) as previously described (Ojo et al., 2021 (link)). Membranes were incubated overnight at 4°C in primary antibodies: (p‐eNOS Ser1119 [ThermoFisher, #PIPA564613]; NOS3 [ThermoFisher, #4904]; p‐IKB [Santa Cruz Biotechnology, Dallas, TX, #SC‐8404]; PI3K [Santa Cruz Biotechnology, #SC‐365290]; IKB [Santa Cruz Biotechnology, #SC‐1643]; p‐NFkBp65 Ser536 [#sc‐136548, Santa Cruz Biotechnology]; NFkBp65 [Santa Cruz Biotechnology, #SC‐8008]; and GAPDH [#60004–1, Proteintech, Rosemont IL]) diluted in 5% bovine serum albumin (BSA). Membranes were washed in PBS and incubated for 1 h in anti‐rabbit (Cell Signaling Technology, Danvers, MA #7074) or anti‐mouse (Cell Signaling Technology, #7076) IgG HRP‐linked antibody diluted in 5% milk solution. Proteins were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific, #34095). Band signals were captured with the ChemiDoc imaging system (BioRad) and quantified with the Image J software, v 1.8.0.

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).