Figure 3

Reduction of N-cadherin leads to reduction of AKT and LRP6 phosphorylation. (A) Transfection of N-cadherin-shRNA (Ncad-shRNA) reduced the amount of AKT phosphorylated at Serine 473 (P-AKT [S473P]) and AKT targets β-catenin phospho-Serine 552, β-catenin [S552-P]) and phosphorylated GSK3β (P-GSK3β [Ser 9]). N-cadherin, P = 0.0151 by paired t-test, n = 7; P-AKT [S473], P = 0.0182 by paired t-test, n = 6; P-β-catenin [S552], P = 0.0083 by paired t-test, n = 3; P-GSK3β [Ser 9], P = 0.0085 by paired t-test, n = 3. Consistent with the stabilizing role of phosphorylated Ser 552, there was a trend for reduction in total β-catenin in N-cad-shRNA transfected cells (P = 0.1383 by paired t-test, n = 2). (B) Cells stably expressing N-cad-shRNA knockdown reveal reduced levels of AKT phosphorylation (P-AKT [S473], phospho-GSK3β (P-GSK3β [S9]), and phospho-β-catenin (P-β-catenin [S552]). N-cadherin knockdown also reduced baseline phosphorylation of LRP-6 (P-LRP6 [S1490]) as well as Wnt3a-induced phosphorylation of LRP6. (C) Primary mouse cortical progenitors were nucleofected with two different shRNA to N-cadherin or EGFP and cell extracts were blotted for phospho-β-catenin Serine 552 (P-β-catenin [S552]). In both cases, N-cadherin knockdown resulted in reduced phosphorylation of β-catenin at Ser 552. shRNA1: P = 0.0119 by paired t-test (n = 3); shRNA2: P = 0.0055 by paired t-test (n = 3). (D) Primary mouse neural stem cells derived from E14.5 mouse cortices were treated for 24 hours with either control IgG or N-cadherin function blocking antibody 20 µg/ml, and cell extracts were blotted for phospho-LRP6 [S1490]. Both biological replicates are shown.