Anti hur mouse monoclonal antibody

Retinal tissues were homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.25% Na-deoxycholate, 1% Nonidet, and a protease inhibitor cocktail) in a glassteflon homogenizer, sonicated (10 sec for 2 times), and centrifuged (980 x g for 3 min at 4°C). The pellet was discarded; the protein content in the surnatant was measured according to Bradford's method using bovine albumin as a standard. Proteins of interest were analyzed by Western blotting or ELISA following standard procedures. For Western blotting, the anti-HuR mouse monoclonal antibody and the anti-α-tubulin rat monoclonal antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted at 1:1000 in TBST buffer [10 mM Tris-HCl, 100 mM NaCl, 0.1% (v/v) Tween 20, pH 7.5] containing 6% milk. The nitrocellulose membranes were processed using Pierce ECL Plus from Thermo Scientific (Rockford, IL, USA). α-tubulin was used as housekeeping to normalize HuR data.
Densitometric analysis was performed using the NIH Image software (http://rsb.info.nih.gov/nih-image). VEGF protein level was measured via an ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) recognizing VEGF-A isoforms of 120, 164, and 188 amino acids, according to our previous publication [13] . Masked evaluation of HuR and VEGF levels was performed.