Primary monoclonal antibody against β actin

Total protein in aortic tissues were washed twice with ice-cold PBS and solubilized in buffer A [5 mM Tris-Cl, pH 7.5, containing 2 mM dithiothreitol (DTT), 2 mM EDTA, 1 mM EGTA, 5 lg/mL each of leupeptin, aprotinin, pepstatin A and chymostatin, 50 mM potassium fluoride, 50 nM okadaic acid, 5 mM sodium pyrophosphate, and 2% sodium dodecyl sulfate (SDS)] and sonicated to dissolve the tissue completely. BCA kit (Pierce Company, Rockford, IL, USA) was used to determine the protein concentration. Proteins (40 μg) from each sample were loaded on 10% SDS-polyacrylamide gel electrophoresis. The gels were electrophoresed, and then transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare) at 4 °C. After rinsed with TTBS (20 mM Tris-Cl, pH 7.5, 0.15 M NaCl and 0.05% Tween-20), the transferred PVDF membrane was blocked with 10% skim milk in TTBS for 1 h and incubated with the CSE, TGF-β, p-Smad3 and total-Smad3 (Santa Cruz Biotechnology, Inc., CA 95060, USA, 1:1000 dilution) for 3 h. The membrane was washed and incubated with IRDye® 800CW Goat anti-Rabbit IgG or Goat anti-Mouse IgG secondary antibody (1:5000 dilution) at room temperature for 2 h followed by LiCor Odyssey gel imaging scanner detection (LI-COR Biosciences, Lincoln, NE). To verify equal loading of protein, the blots were reprobed with primary monoclonal antibody against β-actin (ProteinTech Company, USA).

Total protein was extracted from the L4 and L5 DRGs using methods described earlier [15 (link)]. The primary antibodies against pCaMKIV, CaMKIV, HMGB1 (1:500; Santa Cruz, USA) were used in Western blot (WB). To verify equal loading of protein, the blots were reprobed with primary monoclonal antibody against β-actin (ProteinTech Company, USA).