Horseradish peroxidase conjugated anti rabbit or anti mouse igg

Protein extracts (20 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4-12 % gel, transferred to nitrocellulose membranes, and blocked for 1 hour at room temperature in Blocking One (Nacalai Tesque, Tokyo, Japan). The membranes were incubated with rabbit anti-ADORA2B polyclonal antibody (Chemicon International, Temecula, CA, USA), mouse anti-α tubulin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti- HIF-1α monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-Akt monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-pAkt monoclonal antibody (Santa Cruz Biotechnology) , rabbit anti-ERK1/2 monoclonal antibody (Santa Cruz Biotechnology), and rabbit anti-pERK1/2 monoclonal antibody (Santa Cruz Biotechnology) overnight at 4 °C. The membranes were washed with 0.1 % Tween-20 in Tris-buffered saline, incubated with secondary antibody and coupled to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI, USA) for 1 hour at room temperature. The membranes were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo), and immunoblotting was visualized by exposing the membranes to ATTO Light-Capture II (ATTO, Tokyo, Japan). Signal intensities were quantitated using the CS Analyzer version 3.0 software (ATTO).

The cells were lysed in RIPA buffer (10 mM Tris-HCL, 1 mM EDTA, 1% sodium dodecylsulfate (SDS), 1% NP-40, 1:100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The samples were separated on a 10% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA,USA). The membranes were blotted with 5% dehydrated milk for 2 h and then incubated with primary antibodies overnight. The membranes then were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI, USA) and visualized using SuperSignal reagents (Pierce, Rockford, IL, USA). The primary antibodies used were anti-HA (Clone No. C29F4, Cat No. 3724, Cell Signaling Technology, Beverly, MA, USA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Clone No. GAPDH-71.1, Cat No. G8795, Sigma-Aldrich), which is a housekeeping gene.

MSCs were lysed in RIPA buffer (10 mM Tris–HCl, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 1% NP-40, 1: 100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The samples were separated on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer apparatus (Bio-Rad). The membranes were blotted with 5% dehydrated milk for 2 h and then incubated with primary antibodies overnight. The immune complexes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, USA) and visualized with SuperSignal reagents (Pierce, USA). Primary polyclonal antibodies against RBPJK, phosphor-smad1/5/8, phospho-ERK1/2, phospho-p38MAPK, total-ERK1/2 and total-p38MAPK (Cell Signaling, USA) were used. We also used a primary monoclonal antibody to detect the housekeeping protein, β-actin (Sigma-Aldrich, USA).