Polyvinylidene membrane

T-MSCs and T-MSC-SCs were washed with ice-cold PBS and lysed in PRO-PREP buffer containing a phosphatase inhibitor cocktail solution (iNtRON Biotechnology, Seongnam-si, Korea) for 30 min on ice. After centrifugation at 13,000× g for 20 min at 4 °C, equal quantities of protein from supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and were electrophoretically transferred onto polyvinylidene membranes (Millipore, Billerica, MA, USA). The blots were then probed overnight at 4 °C with antibody against the glial fibrillary acidic protein (GFAP) (1:400, monoclonal antibody, Sigma-Aldrich, cat. no. G3893) or the nerve growth factor receptor (NGFR/p75) (1:500, polyclonal antibody, Santa Cruz Biotechnology, cat. no. sc8317, Dallas, TX, USA), followed by the corresponding secondary antibody. The blots were washed and developed using enhanced chemiluminescence reagents (WestSave GOLD™ Western Blot Detection kits) (AbFrontier, Seoul, Korea), according to the manufacturer’s instructions. Band intensities were assessed by densitometric scanning (LAS-3000, Fujifilm, Tokyo, Japan).

The tissue samples were washed twice with phosphate buffered saline (PBS), homogenized, and lysed in the RIPA buffer (Upstate, Billerica, MA) for 20 min at 4 °C. Lysates were centrifuged at 12,000 g at 4°C for 12 min and we separated supernatants for western analysis. SDS-PAGE (10% gel) was used to separated equal amount (40 mg) of cell lysates, followed by transferring them to polyvinylidene membranes (Millipore, Billerica, MA), and then probed them with antibodies against proteins of interest, including anti-Heme oxygenase1 antibody (1:1000, room temperature for 2 h, Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin antibody (1:10000, room temperature for 1 h, Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate horse radish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were adopted to incubate blots. Enhanced chemiluminescence (ECK) kit (Amersham ECL detection system, GE Healthcare) was used to visualize bound antibodies, and the relative density of the target bands was determined by using densitometry analysis.

Equal amounts of proteins were separated by SDS-PAGE, and then transferred onto polyvinylidene membranes (Millipore, SaintQuentin enYvelines, Belgium) by electrotransfer. Membranes were blocked with 5% skim milk in PBS-T (containing 0.1% Tween-20), and proteins of interest were visualized using specific Anti-Stathmin (Cell SignalingTechnology, Beverly, MA, USA), poly (ADP-ribose) polymerase (PARP) (BD Biosciences, San Jose, CA, U.S.A.), integrin β1, and integrin α5(BD Biosciences, San Jose, CA), caspase-3 (Santa CruzBiotech, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody and secondary antibodies, conjugated with horseradish peroxidase (HRP), were ordered from KangChen Biotech (Shanghai, China).
For immunoprecipitation, the cells were lysed inbuffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet™ P-40 (NP-40), 5 mM EDTA, 5 mM ethylene glycol tetraacetic acid, 15 mM MgCl2, 60 mM β-glycerolphosphate, 0.1 mM sodiumorthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF. Twenty microliters of protein A/Gagarose beads (BD Bioscience Pharmingen) were added to the lysates for proper periods of incubation. The beads were then washed and subjected to SDS-PAGE and immunoblotting.