Research Article

Generation of Constitutive Active ERK Mutants as Tools for Cancer Research in Zebrafish

Figure 3

In vivo ERK signaling activity analysis of ectopically expressed FGF or ERK isoforms.(a) western blot detection of dpERK on protein samples isolations from 5 hpf embryos injected with 50 pg WT or mutant ERK2 mRNA and isolated. (b) Flowchart of the experimental procedure using the Tg(Dusp6:d2EGFP) and subsequent COPAS .analysis (c). 5 hpf old living embryos are passed through the COPAS flowcell and optical density/axial length (blue), GFP (green), YFP (yellow), and dsRED (red) fluorescence profiles are recorded Union Biometrica, Inc. D. Peak height values (PH) of the GFP channel were collected for ERK2_WT, , , and FGF8 mRNA injected embryos, after corrected for autofluorescence/background of noninjected wild-type embryos (see Section 2), and plotted in a bar graph as the mean value of three biological experiments, each with a minimum of 30 embryos/condition. Mean GFP PH values are normalized against ERK2_WT. Arrow bares represent standard deviations of the means of the three biological experiments; P values were calculated by applying a t-test with Welch-correction.