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. 2017 Feb 17;27(3):1732–1747. doi: 10.1093/cercor/bhx023

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© The Author 2017. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — JAK2 and SH2B1β are necessary for KIAA0319-induced activation of Smad2 and inhibition of axon growth. (A) Western blot analysis of SH2B1β levels in CAD cells transfected with different shRNA against SH2B1 (shSH2B1#1, upper panel; and shSH2B1#2, lower panel) or a control plasmid (shControl) following puromycin selection. (B) Quantification of A. (C) Western blot analysis of pSmad2 and total Smad2 levels in CAD cells depleted of SH2B1 (shSH2B1#1, upper panel and shSH2B1#2, lower panel) or transfected with a control shRNA plasmid (shControl) overexpressing either KIAA0319 (hKA) or an empty vector (control). (D) Quantification of C. (E) Western blot analysis of pSmad2 and Smad2 levels in JAK2-deficient cells (gamma-2-A; γ2A) and in the control parental cell line (2C4) upon overexpression of KIAA0319 (hKA) or an empty vector (control). (F) Quantification of E. (G) Western blot analysis of JAK2 levels in CAD cells transfected with different shRNA against JAK2 (shJAK2#1, upper panel; shJAK2#2 and shJAK2#3, lower panel) or a control shRNA, following puromycin selection. (H) Quantification of G. (I) Representative photomicrographs of anti-βIII-tubulin immunofluorescence in differentiated GFP-positive CAD cells (highlighted with an arrowhed) stably transfected with either a control shRNA plasmid (shControl), a shRNA against SH2B1 (shSH2B1#1), or a shRNA against JAK2 (shJAK2#1) and overexpressing either KIAA0319 (hKA) or an empty vector (control). Scale bar, 50 μm. (J) Quantification of total neurite length of I; shControl (control: n = 162; hKA: n = 152 cells), shSH2B1#1 (control: n = 112; hKA: n = 72 cells), shSH2B1#2 (control: n = 201; hKA: n = 149 cells), shJAK2#1 (control: n = 126; hKA: n = 89 cells), shJAK2#2 (control: n = 136; hKA: n = 167 cells) and shJAK2#3 (control: n = 80; hKA: n = 87 cells). (K) Quantification of axon length of hippocampal neurons transfected with both hKA and different shRNAs- shControl (control: n = 188; hKA: n = 67 cells), a shRNA against SH2B1 (shSH2B1#1; control: n = 78; hKA: n = 56 cells) or a shRNA against JAK2 (shJAK2#1; control: n = 32; hKA: n = 81 cells). Results are expressed in mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = not statistically significant.