Fig. 3

Knock-down of fak1a and fak1b phenocopies the Paxillin morphant phenotype.

(A) Western blot analysis of FAK protein levels in Paxillin-deficient zebrafish embryos compared to control-injected embryos. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. The figure shows one representative western blot out of three independent experiments. (B, C) Quantitative real time PCR of Paxillin-depleted and control-injected embryos showed no significant (ns) alteration of fak1a (n = 3; *P = 0.9017) (B) and fak1b (n = 3; *P = 0.8878) (C) mRNA expression. For statistical analysis student’s t-test was performed. (D-F) Lateral view of MO1-fak1a/fak1b (D) and 5bp-mismatch-MOs (MO1-control) (E) injected embryos at 72 hpf. Embryos co-injected with 2.7 ng of MO1-fak1a and 2.15 ng of MO1-fak1b showed a severe heart failure phenotype whereas injection of the same amount of fak1a and fak1b 5bp-mismatch MOs (MO1-control) caused no significant pathological cardiac phenotype (F) (n = 3; *P<0.0001). (G) FS measurements of FAK morphant ventricles at 36, 48 and 72 hpf. FS of FAK double-knock-down morphant ventricles was slightly reduced to 56.38% ± 5.85% compared to corresponding 5bp-mismatch-MO injected embryos (FS: 62.4% ± 5.77%) at 36 hpf. At 48 hpf, FS in FAK morphants was reduced to 63.57% ± 9.18% compared to control morphants (70.33% ± 3.72%), whereas ventricular chambers of FAK-deficient embryos became almost silent (FS: 2.6% ± 3.71%) compared to controls (FS: 69.33% ± 4.36%) at 72 hpf (n = 5–8 individuals per time point). (H) Western blot analysis of Paxillin levels in control-, MO1-fak1a/fak1b- and MO1-paxillin-injected embryos. Paxillin protein levels are severely reduced after targeted knock-down of FAK and Paxillin, respectively. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. The figure shows one representative western blot out of three independent experiments. (I) Quantitative real time PCR of FAK-depleted and control-injected embryos showed no significant (ns) alteration of paxillin mRNA expression (n = 3; *P = 0.0937). For statistical analysis student’s t-test was performed. (J) Western blot analysis of embryos co-injected with zebrafish paxillin mRNA and MO2-paxillin compared with embryos injected with MO2-paxillin or paxillin mRNA alone. Ectopic expression of zebrafish paxillin mRNA was able to restore FAK protein expression. For each sample 50 embryos were pooled and 20 μg of protein lysate were loaded per lane. Error bars indicate s.d.