Immobilon p polyvinylidene difluoride membranes

Cells were homogenized in a NP-40 lysis buffer containing protease and phosphatase inhibitors and protein concentration was determined using the BCA method. Homogenates were centrifuged for 15 min at 13,000 xg at 4°C and supernatants were collected. Protein samples (40 μg) were separated onto 8 or 10% reducing polyacrylamide gels and electroblotted onto Immobilon-P polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). Immunoblots were blocked either 5% milk in Tris-buffered saline or with Blocking Buffer for Fluorescent Western Blotting (Rockland Antibodies & Assays, Gilbertsville, PA, USA) for 2 h at room temperature and incubated overnight at 4°C with the indicated antibodies in Tris-buffered saline and 0.05% Tween 20 (TBST) containing 1% BSA. Membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:30,000) for 45 min at room temperature. Membranes were washed in TBST, and antigen-antibody complexes were visualized by chemiluminescence using an ECL kit (Pierce, Rockford, IL, USA). Alternatively, immunoblots were incubated with IRDye800CW Goat Anti Mouse (H+L) or IRDye680 Goat Anti Rabbit (H+L) secondary antibodies and signal was detected with the Odyssey® Infrared Imaging System (Li-COR Biotechnology, Lincoln, NE, USA). The relative band intensity was quantified using the ImageJ software.

Total eNOS and vimentin protein expression was evaluated by western blot in hWMSCs, hWMSC-End14d or hWMSC-End30d. Total proteins (50 µg) were separated by a polyacrylamide gel (10%) electrophoresis and transferred to Immobilon-P polyvinylidenedifluoride membranes (BioRad Laboratories, Hertfordshire, UK). Membranes were probed with rabbit anti-eNOS (1∶1000), anti-vimentin (1∶500) or anti-β actin (1∶2000) (Santa Cruz Biotechnology, CA, USA). Membranes were incubated (1 h) with a horseradish peroxidase-conjugated goat anti-rabbit antibody. Proteins were detected by the enhanced chemiluminescence method, analyzed by densitometry and compared to β-actin expression (control).