Figure 2
From: Nuclear connectin novex-3 promotes proliferation of hypoxic foetal cardiomyocytes
Novex-3 nuclear localisation appears to be regulated by the nuclear localisation signal (NLS) in the N-terminal region. (a) Primary structure of human novex-3 (assembled from refs20,36). Mouse novex-3 has a similar molecular structure (Supplementary Fig. S9). The epitope of the two different antibodies used in this study is marked with “e1” and “e2”, both of which is within the novex-3-specific exon (see Methods). The nuclear localisation signal (NLS) located at N-terminus between Z2 and Z repeats17 is shown. (b) Four fragments covering the full length novex-3 (novex-3-1 through novex-3–4) as well as novex-3-Nterm fragment containing connectin exons 1 to 6 (shown in (a)), were independently expressed as N-terminal EGFP-tagged proteins in cultured fCMs (E16–E18) by baculovirus-mediated protein expression system. Live cells were observed by fluorescence microscopy together with bright-field optics. The approximate protein size of these five fragments, excluding the tagged-EGFP (~27 kDa), was 146, 206, 170, 146 and 32.6 kDa for novex-3–1, novex-3-2, novex-3-3, novex-3–4 and novex-3Nterm, respectively. (c) The E16 fCMs doubly expressing novex-3-Nterm-GFP and sarcomeric α-actinin-mCherry (left), or novex-3-1-GFP and sarcomeric α-actinin-mCherry (right), were shown together with bright-field images. Sarcomeric α-actinin was used as a specific CM marker. Scale bars = 20 µm in (b) and 30 µm in (c).
