Supplementary Figure 1: Protein expression levels are unchanged in constitutive Nrxn3α/β KO brains.
a. Representative Western blots - cropped images for 25 neuronal proteins in brain homogenates harvested from three sets of adult littermate wild-type and Nrxn3a/b KO mice.
b. Quantification of protein concentrations in adult wild-type and Nrxn3α/β KO brains (n=3 mice each).
c. qRT-PCR from mRNA isolated from wild-type, Nrxn3α+/– and Nrxn3α–/– brains and probed for transcripts representing Nrxn3α (left) and Nrxn3β (right). Note that based on the KO strategy (Fig. 1), the 5’ end of the Nrxn3α mRNA is still synthesized, but the resulting RNA is likely to nonsense-mediated decay resulting in a reduced level of mRNA for Nrxn3α. If the low levels of truncated mRNA produced a stable protein despite the presence of a partial C-terminal LNS-domain that is unable to fold due to the truncation, the produced protein would be secreted because it has no membrane anchor, and thus would be non-functional in all assays we have performed (see Fig. 7 this paper, and Aoto et al., 2013; Nrxn3α expression: Nrxn3α/β+/– vs. Nrxn3α/β –/–, p < 0.0001; Nrxn3β expression: Nrxn3α/β+/– vs. Nrxn3α/β –/–, p < 0.0001).
All data are shown as percent of control. Protein concentration levels were determined by phosphorimager analysis using I125 conjugated secondary antibodies. Data are means ± SEM. *, p<0.05 by Student’s t test comparing genotype protein levels and single factor ANOVA compared qRT-PCR.
