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Fig. 7 | BMC Neuroscience

Fig. 7

From: Downstream mediators of Ten-m3 signalling in the developing visual pathway

Fig. 7

Ten-m3 interacts with the Zic2–EphB1 signalling pathway. a Western blot showing immunodetection of a 60 kDa band corresponding to the expected size of Zic2 following GST pull-down assay with mouse P1 whole brain lysates; sepharose 4B beads (lane 1), GST alone (lane 2), Ten-m3-GST fusion protein probe (lane 3), and supernatants of beads only (lane 4), GST alone(lane 5), and Ten-m3-GST (lane 6). The 60 kDa Zic2 band can be observed in the lane containing Ten-m3-GST (lane 3) but not beads (lane 1) or GST alone (lane 2). Additionally, immunoreactivity for Zic2 can be seen in all supernatants (lanes 4–6) confirming that Zic2 was present and suggesting the Ten-m3-GST probe concentration was unable to bind all Zic2 present in the lysate. b As for a following repeat pull-downs using lysate from three individual animals. Lane 1 GST alone; Lane 2 Ten-m3-GST fusion protein probe. These findings confirm a reliable and specific interaction between Zic2 and the intracellular domain of Ten-m3. c Realtime qPCR revealed significant up-regulation of Zic2 in retina (1.5 fold, p < 0.001), and SC (1.7 fold, p = 0.001) in Ten-m3 KOs compared to WTs. These results suggest that Ten-m3 may modulate the expression of Zic2 by repressing transcription. A correlated increase in EphB1 mRNA was also observed both in the retina (fold change 2.25 ± 0.13; p = 0.016) and SC (fold change 2.04 ± 0.5; p = 0.011). Graph shows relative fold change ± 1SE, normalised to Gapdh and in comparison to WT controls. Statistical significance is denoted by (*): p < 0.05*, p < 0.01**, p < 0.001***

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