Figure 5

The N-terminal domain of VDAC2 contains critical elements for its cardioprotective activity. (A) Schematic representation of VDAC2/3 chimeric constructs. Reciprocal chimeric constructs were generated by swapping the N- and C-terminal halves of VDAC2 and VDAC3 and tagging a FLAG epitope at the C-terminal end. VDAC^N2C3: The N-terminal half of VDAC2 (amino acids 1 to 142) was fused to the C-terminal half of VDAC3. VDAC^N3C2: The N-terminal half of VDAC3 was fused to the C-terminal half of VDAC2. (B) Western blot with anti-FLAG antibody after SDS-PAGE with lysates from 30 hpf uninjected embryos or embryos injected with 60 pg. FLAG-tagged chimeric VDAC mRNA. β-actin was used as a loading control. (C) Injection of VDAC^N2C3 mRNA restored synchronized cardiac contractions in 1-day-old tremblor/ncx1h embryos (43.4 ± 2.8%, N = 3, n = 168 compared to 13.7 ± 3.1%, N = 3, n = 156 in uninjected siblings), whereas injection of VDAC^N3C2 mRNA fails to produce this effect (14.2 ± 1.2%, N = 3, n = 162). Overall rescue percentages represent the mean rescue percentage ± s.e.m. from N independent experiments, using a total of n embryos.