Validation of CREBBP-methylated specific antibodies on zebrafish embryos. (A,B) Immunohistochemistry with CREBBPmethylated specific antibody at 18 ss using (A) R714me2a or (B) R742me2a antibody on embryos injected with Control morpholino (CTRMo) and CREBBPb morpholino (CREBBPbMo). Nuclei are stained in blue with To-Pro3 and a merged picture is shown. Expression of CREBBP-R714me2a and CREBBP-R742me2a is strongly reduced in CREBBPbMo- as compared to controlMo-injected embryos. Injections were performed at the 1-cell stage with 6 ng of CTRMo or CREBBPbMo (Gene-tools). Morpholino sequences were: CREBBPb 5′-GACTGTTGCCACCTGCCATGCCCAT-3′ and Control 5′-CCTCTTACCTCAGTTACAATTTATA-3′. (C) Knock-down of CREBBPb reduces CREBBPb protein level in 14 ss old zebrafish embryos. Embryos injected with either CTRMo or CREBBPbMo were collected at 14 ss and processed for western blot analysis to detect CREBBP expression. Tubulin was used as a loading control and a protein ladder is shown. Whole cell extracts from 20 embryos were classically prepared in 40 ml Laemmli sample buffer. 5 ml of each sample (4 embryos) were loaded per lane and subjected to SDS-PAGE analysis. The following antibodies were used: anti-CREBBP (1/200, A22, santa Cruz), anti a-Tubulin (1:5000, Sigma, T9026), anti-Rabbit IgG, HRP conjugate (1:50000, Promega, W4011) and anti-Mouse IgG, HRP conjugate (1:10000, Promega, W4021). Protein Ladder, PageRuler prestained Protein Ladder (Fermentas, SM0671).