Enhanced chemi luminescence (ecl)

Western blotting was performed, as previously described,32 (link) to confirm Dicer overexpression and the level of VEGF-A. Equal protein quantities from total cell lysates were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. Membranes were blocked for 2 h in blocking buffer (5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20) at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-Dicer (dilution 1:1000; Abcam, Cambridge, UK), anti-VEGF-A (dilution 1:1000; Abcam, Cambridge, UK) and anti-β-actin (dilution 1:5000; Abcam, Cambridge, UK), followed by incubation with an anti-mouse IgG antibody (Abcam, Cambridge, UK) at a dilution of 1:5000. The relative intensities of protein bands were visualized using ECL (BD, San Diego, CA).