Figure 1.
Co-localization of LRRTM3 and a) APP in early endosomes, b) BACE1 in early endosomes, c) APP in primary neurons.
For a and b, SH-SY5Y-APP695wt cells were transduced with baculovirus expressing fused early-endosomal protein Rab5a and GFP. These cells were transfected with LRRTM3-V5 (a and b) and also with BACE1-HA (b). Results of staining with GFP fluorescence indicative of Rab5a expression (early endosomes, green); anti-V5 (LRRTM3, red); and either CT20 in a (APP, magenta) or anti-HA in b (BACE1, magenta) are shown with overlay of the three stains in the last panels of a and b. Co-localization of APP, LRRTM3 and early endosomes is visualized as white punctate intracellular structures in the last panel of a(arrow and arrowhead). Co-localization of APP, BACE1 and early endosomes is visualized as white punctate intracellular structures in the last panel of b. (Magnification: ×63). For c, primary neuronal cultures from Tg2576 transgenic mice transduced with rAAV-LRRTM3-V5 were stained with anti-V5 (LRRTM3, green) and CT20 (APP, red). Overlay of the two stains reveals co-localization of LRRTM3 and APP visualized as yellow puncta in the cell body (thin arrow) and neuronal process (thick arrow). (Magnification: ×100).
Figure 2.
Co-IP of BACE1 and endogenous APP with LRRTM3 in HEK293T cells.
LRRTM3-V5 and BACE1-GFP were transfected into HEK293T cells as shown in (a) and as in inputs (b). Protein lysates from these cells and negative controls without overexpression were immunoprecipitated using c. anti-V5 (LRRTM3), d. anti-GFP (BACE1) or c. CT20 (APP) antibodies. In the IP with anti-V5 (c), Western blot assays using anti-GFP or CT20, demonstrate co-IP of BACE1 and endogenous APP with LRRTM3. Similarly, LRRTM3 staining is clearly demonstrated in IPs for BACE1 (d) and for APP (e), indicating reverse co-IP of LRRTM3 with both proteins. Presence (+) or absence (−) of each of the three proteins for the depicted experiments are shown in (a) for each of the experimental conditions within the four lanes. Antibodies used in the Western blots are listed to the right of each figure. Proteins that are IP’ed or co-IP’ed are shown to the left of each figure.
Figure 3.
Relative expression levels of genes in H4 cells treated with three anti-LRRTM3 and a control siRNA.
Bar graphs depicting mean relative gene expression levels and error bars representing the standard deviations obtained from the averages of 2–6 experiments where each experiment is assessed in quadruplicate. Relative expression values are obtained by the delta delta Ct method, where HPRT is utilized as the control gene (delta Ct) and all results are normalized to one of the control wells (delta delta Ct). Relative expression values (2?(-delta delta Ct)) are plotted on the y-axis. The different siRNA treatment groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.
Table 1.
Human brain expression correlations between levels of LRRTM3, and CTNNA3, BACE1 or APP.
Figure 4.
Expression levels of genes in brains of Lrrtm3 knock-out, heterozygote and wild type mice.
Bar graphs depicting mean gene expression levels and error bars representing the standard deviations obtained from the averages of 3 animals per genotypic group where expression levels from each mouse brain is assessed in quadruplicate. Expression values are obtained by the delta Ct method, where geometric mean of HPRT and GAPDH is utilized as the control gene expression values. Average expression values (2?(-delta Ct))x100 were plotted on the y-axis. The three mouse genotypic groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.
Table 2.
Multilocus genotype (MLG) analysis results.