Fig. 4

TCF3 is a target of ASB2 in cardiomyocytes.

(a) Relative mRNA expression (qPCR) of socs3b, id2a and mef2cb in isolated 50 hpf WT (blue bar) and asb2b mutant (orange bar) hearts (n=2 technical replicates; RNA samples obtained from 500 isolated hearts). (b) Co-immunoprecipitation (IP) of HA-ASB2? and Myc-E12 or Myc-E47 immunoblotted (IB) for HA (arrow) or Myc. Rat NCMs were transfected with HA-Asb2? or control (mock), together with Myc-E12 or Myc-E47 adenovirus vectors. All adenovirus vectors used contain an independent GFP cassette to assess transfection efficiency. After 24?h of transfection, cells were treated with 20??M MG132 for 8?h and then IP was performed; lower bands in HA blot are HA antibody heavy chains. (c) Asb2? induced E12 and E47 degradation in a proteasome-dependent manner. Rat NCMs were transfected with control, HA-Asb2? or HA-Asb2? L595A mutant (Asb2?LA), together with Myc-E12 or Myc-E47 adenovirus vectors. After 48?h of transfection, NCMs were treated with 20 uM MG132 or DMSO for 12?h and these protein samples were extracted for immunoblotting. (d) Three-dimensional images of a 50 hpf TgBAC(asb2b:GFP-asb2b);Tg(myl7:MKATE-CAAX) atrium, and a Tg(myl7:MKATE-CAAX) atrium expressing EGFP-Tcf3b. Both Asb2b and Tcf3 localize to the nucleus in cardiomyocytes. (e) Three-dimensional images of 50 hpf Tg(myl7:LIFEACT-GFP) and Tg(myl7:LIFEACT-GFP);Tg(myl7:EGFP-tcf3b-2A-tdTomato) atria. (The transgenic fish exhibit mosaic expression of EGFP-tcf3b-2A-tdTomato in the heart.) EGFP-Tcf3b-2A-tdTomato expressing cardiomyocytes exhibit disassembly of myofilaments. Arrow points to a severe case of myofilament disassembly, arrowhead to a mild case. Number of cardiomyocytes exhibiting sarcomere disassembly (n=5 hearts, with averages taken from 20 cardiomyocytes per heart). (f) Three-dimensional images of 50 hpf Tg(myl7:ras-GFP) or Tg(myl7:ras-GFP);Tg(myl7:GFP-tcf3b-2A-tdTomato) atria. Tcf3b-2A-tdTomato expressing cardiomyocytes exhibit abnormal membrane protrusions (arrows). Number of cardiomyocytes exhibiting membrane protrusions (n=5 hearts, with averages taken from 20 cardiomyocytes per heart). (g) Quantification of myofilament branch points, cellular morphology and membrane protrusions in 50 hpf WT, asb2b mutant, Tg(myl7:GFP-tcf3b-2A-tdTomato) WT and Tg(myl7:id2b-2A-tdTomato) asb2b mutant atrial cardiomyocytes in Tg(myl7:LIFEACT-GFP) or Tg(myl7:ras-GFP) background. (n=15 to 34 cardiomyocytes from 3 to 6 hearts). *P<0.05 and **P<0.01 by one-way analysis of variance (ANOVA) followed by Tukey?s honest significant difference test. Error bars, s.e.m. Scale bars, 20??m.