Rabbit anti flag antibody

Secreted proteins were collected from the VEGF/hUCB‐MSCs and hUCB‐MSCs. After culturing the cells in growth medium for 3 days, the cells were washed five times with serum‐free media and incubated with serum‐free media for 24 hours. Proteins in the conditioned media were precipitated with 10% (vol/vol) trichloroacetic acid (Sigma‐Aldrich) at 4°C. The protein pellets were air dried prior to use. Western blot analysis was performed as previously described 24. Briefly, hUCB‐MSCs were washed twice with cold PBS and lysed with RIPA buffer containing protease inhibitors (Roche, Basel, Switzerland, http://www.roche.com). For primary antibody labeling, rabbit anti‐flag antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com) and rabbit anti‐human VEGF polyclonal antibody (1:2,000, AbClon, Seoul, Korea, http://abclon.com) were used. An horse radish peroxidase (HRP)‐conjugated anti‐rabbit IgG antibody (1:2,000, Enzo Life Sciences, Farmingdale, NY, http://www.enzolifesciences.com) was used as a secondary antibody.

Lysates from HEK293 cells transfected with different GFP- and FLAG-tagged fusion proteins were incubated for 3 h at 4°C with 6 µg of monoclonal anti-FLAG antibody, 3 µg of rabbit anti-FLAG antibody, mouse anti-GFP antibody, and nonimmune mouse and rabbit IgG (Santa Cruz Biotechnology, Inc.) prebound to protein A/G–agarose (5 h, 4°C; Santa Cruz Biotechnology, Inc.). Lysate preparation, matrix coating, binding, and washing were performed in lysis buffer containing 75 mM NaCl (for syndapin I–ProSAP1 coimmunoprecipitation) and 100 mM NaCl (for syndapin I/syndapin I coimmunoprecipitation). Coimmunoprecipitated proteins were analyzed by immunoblotting using rabbit polyclonal anti-FLAG antibodies, rabbit polyclonal and mouse monoclonal anti-GFP antibodies, and rabbit anti–syndapin I antibodies.
Several independent experiments were conducted to reproduce data.