Anti gfp clone b 2

Lysates (without crosslinking) and beads were prepared as described in the iCLIP procedure. Immunoprecipitation was performed with 500mg protein as described in the iCLIP procedure. Immunoblotting was performed by standard procedures. 50µg protein of crude lysate was loaded and 10% of the unbound fraction or IP, respectively. Antibodies for immunoblotting were: anti-BRF1/2 (Cell Signaling), anti-GFP (clone B2, Santa Cruz), anti-Tubulin (DM1A, Sigma).

Zebrafish embryos were incubated in 5 mg/ml BrdU, 1% DMSO in E3 medium from 48 hpf to 72 hpf at 28°C, rinsed three times i n E3 medium and fixed overnight in 4% PFA. Fixed embryos were rinsed in PBST, bleached in the dark for 20 min (using 0.8% KOH, 0.9% H2O2 and 1% Tween-20 in distilled water), permeabilized using 1% Triton-X100 in PBS for 2 h and equilibrated in DNase I buffer (40 mM Tris-HCl, pH 8.0, 10 mM MgSO4, 1 mM CaCl2) for 30 min at 37°C. Equili brated embryos were treated with DNase I (1:50 in equilibration buffer) for 2 h at 37°C, rinsed three times in PBSTw (PBS+0.1% Tween20) and subjected to immunofluorescent staining with anti-GFP (clone B-2, Santa Cruz Biotechnology; 1:200) and anti-BrdU (clone BMC9318, Roche; 1:100) antibodies. The ventricles of stained embryos were imaged and analyzed as described above. A ventricular proliferation index (number of BrdU+, GFP+ double positive cells divided by the total number of GFP+ cardiomyocytes) was manually calculated for each embryo using ImageJ and averaged.