Figure 6

Mad3's biological activity depends on 5HT. (A) Sensorgram of serotonin binding to GST-Mad3. Data represent the difference between the sample cell (GST-Mad3) and the reference cell (GST). A blank run (buffer only) has been subtracted from all curves. Four different analyte (5HT) concentrations (2, 1.5, 1, 0.8 and 0.4 mM were injected). Injection start and stop are indicated by arrows. The data shown are representative of 3 independent experiments. (B) Embryos were injected at stage 1 with Mad3 WT or Mad3-5mut and scored for left-right phenotype at stage 45. Embryos injected with Mad3 WT presented significant levels of heterotaxia (17%, n = 52, p = 0.001) whereas Mad3-5mut injected embryos presented only 2% of heterotaxia (n = 104, p = 0.7) when compared to control group. (C) Co-Immunoprecipitation assay. Xenopus embryos at the 1 cell stage were injected with Mad3 WT-flag (lanes 1,2,3) or Mad3-5mut-flag (lanes 4,5,6) and collected at the stage 7 (64 cells). The whole protein lysates were incubated with anti-5HT antibody followed by protein-A agarose. Lane 1: 10% Input; Lane 2: whole lysate from Mad3 WT-flag injected embryos plus anti-5HT; Lane 3: negative control (whole lysate from Mad3 WT injected embryos plus rabbit IgG); Lane 4: 10% input; Lane 5: whole lysate from Mad3-5mut-flag injected embryos plus anti-5HTLane 6: negative control (whole lysate from Mad3-5mut-flag plus rabbit IgG).