Figure 7.
Expression of Csx/Nkx2.5(I183P) and endogenous Csx/Nkx2.5 in TG mice. (a) Immunohistochemistry showed Csx/Nkx2.5(I183P) mutant protein expression in atria and ventricle in embryonic, neonatal, and adult atria (#25-A) and ventricles (#25-V). Bars = 50 mm. (b–g) Adult heart from NTG (b, d, f) and TG (c, e, g) mice were coimmunostained with anti-Csx/Nkx2.5 Ab (d, e, FITC) and anti-FLAG Ab (f, g, rhodamine). Nuclear staining was shown in Blue (b, c). Most of the FITC and rhodamine stainings in e and g were colocalized. Bars = 50 mm. (h) Mutant protein expression at 14 (lane 2) and 17 dpc (lane 4) by Western blotting using anti-FLAG pAb (upper panels) and anti-Csx/Nkx2.5 mAb (lower panels). In lane 2 and 4, both endogenous and mutant proteins were recognized with anti-Csx/Nkx2.5 Ab and showed approximately twofold higher Csx/Nkx2.5 protein expression than NTG (lane 1 vs. lane 2, lane 3 vs. lane 4). (i) Western blot analysis of heart lysate from neonate, 3 and 6 weeks of NTG and TG mice with anti-Csx/Nkx2.5 mAb detected endogenous protein in NTG hearts (lanes 1, 3, 5) as well as the endogenous plus the mutant protein in TG heart (lanes 2, 4, 6). (j) Northern blot analysis of Csx/Nkx2.5 and SV40 poly A. At neonatal stage, Csx/Nkx2.5 mRNA was detected as a major single band in NTG heart (Csx/Nkx2.5, lane 1), and two bands in TG heart (lane 2). The slower migrating ban hybridized with SV40 poly A probe indicating the transcript of I183P mutant (SV40 pA, lane 2). In NTG hearts, Csx/Nkx2.5 mRNA level was downregulated after birth (compare lane 1 vs. lanes 3, 5, 7). However, the downregulation of the endogenous Csx/Nkx2.5 was not observed in TG hearts (lanes 2, 4, 6, 8), indicating the upregulation of endogenous Csx/Nkx2.5 in TG hearts.