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. 2018 Dec 10;28(8):1260–1273. doi: 10.1093/hmg/ddy420

Figure 3.

Figure 3

Expression of HA-NRG1 type III does not affect myelin gene expression. (A) Western blot analyses from WT, Q215X, Nrg1t3;Q215X and Nrg1t3 spinal cord at P30. Blots were probed for HA tag. HA is tagged to the NRG1 type III transgene. Calnexin was used as a loading control. We detected exogenous neuregulin precursor protein level and its cleavage products. The upper band corresponds to the full-length NRG1 type III inactive isoform (135 kDa, arrow); the 75-kDa band corresponds to the active form (arrow head) of NRG1 type III produced by BACE1-cleavage, as described in the literature (30,48). (B, C, F) Western blot and (D) densitometries analyses from WT, Q215X, Nrg1t3;Q215X and Nrg1t3 sciatic nerves at P30. Blots were probed for P-ErbB2, ErbB2 (B), P0, MBP, PMP22, MAG (C) and EGR2 (F). Calnexin and HSP90 were used as loading controls. Protein levels remain steady in all genotypes. (E) Immunogold labeling of P0 protein from cross sections of WT, Nrg1t3, Q215X and Nrg1t3;Q215X sciatic nerves at 1 month. Quantification of gold particles associated with P0 from cross sections of sciatic nerve at P30. Scale bar, 200 nm. (G) Relative quantification of luciferase reporter gene mRNA (Lucif) from Luc D hemizygotes (Luc D), Q215X;LucD, Nrg1t3;Luc D and Nrg1t3;Q215X;LucD sciatic nerves at P15. Luc D mice harbor a combinatorial EGR2/SOX10 binding site upstream of basal Hsp68 promoter. No significant differences were found at P15. Analyses were performed using three mice per genotype. Error bars indicate s.e.m. Statistical analyses were performed using one-way ANOVA with Bonferroni’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.