Fig 2: Knockdown of APP in B103-695 cells results in decreased Ras-MAPK signaling and GSK-3 activity. B103-695 cells were transfected with increasing concentrations of APP siRNA ranging from 30 to 120 nm or with 120 nm scrambled siRNA-A. At 48 h after transfection, cell extracts were prepared and analyzed by Western blot. A, The membrane was initially probed with antibody to 6E10 to visualize knockdown efficiency. The membrane was then sequentially probed with SOS2 (C), Grb2 (E), Ras (G), P-ERK and total ERK (I), CycD1 (K), P-GSK3αβ and total GSK3αβ (M) antibodies. Finally, the membrane was probed with antibody to Actin (O), which served as loading control. The histograms represent the quantitative analysis of protein levels normalized to Actin; full-length APP (B), SOS2 (D), Grb2 (F), Ras (H), and CycD1 (L), respectively. Histograms representing the quantitative analysis of P-ERK and P-GSK3 normalized to corresponding total ERK or GSK3 proteins are also shown (P-ERK [J] and P-GSK3 [N]). Statistical analysis was performed using Student’s t test. The data represent the mean ± SEM from seven independent experiments (n = 7); *p < 0.05.

Fig 3: Effect of PKA activation on downstream WNT pathway phosphorylation.(a) Forskolin represses AXIN2 expression in human adrenocortical H295R cells. Human H295R cells were treated with either vehicle (0, DMSO) or Forskolin 10−5 M (Fsk) for 1 to 24 h and AXIN2 expression was analysed by RT–qPCR. Bars represent mean expression in at least three independent experiments (in triplicate)±s.e.m. Statistical analyses were performed by one-way ANOVA followed by Dunnett's multiple comparison test. (b) Forskolin activates AXIN2 expression in COS7 cells. COS7 cells were treated with Forskolin for 1 to 24 h and AXIN2 expression was analysed as described in a. (c) Differential phosphorylation of β-catenin and GSK3β in COS7 and H295R cells. COS7 and H295R cells were treated as in a. Proteins were extracted and subjected to western blot analysis with antibodies to phospho/dephospho-β-catenin, phospho-GSK3β or the corresponding total proteins. β-catenin phosphorylations/dephosphorylations were sorted as activating (pS552, pS675, active beta-catenin) and inactivating (pT41/S45 and pS33/S37/T41). GSK3β serine 9 phosphorylation (pS9) results in its inactivation. GSK3β tyrosine 216 phosphorylation (pY216) results in its activation. Phospho-GSK3 antibodies recognize phospho-GSK3β arrowheads, and phospho-GSK3α (top band) (d) Phosphorylation of β-catenin and GSK3β in response to constitutive PKA activation in vivo. Activating and inactivating phosphorylations of β-catenin and Gsk3β were analysed in adrenal protein extracts from four wild-type and 4 Sf1:Cre;Prkar1Fl/Fl mice as described in c. (e) Constitutive activation of PKA inhibits activating phosphorylations and stimulates inactivating phosphorylations in vivo. Graphs show pooled quantification of phospho-specific signals in d and Supplementary Fig. 3, representing a total of nine wild-type and nine Sf1:Cre;Prkar1aFl/Fl mice. These were normalized to quantification of total corresponding proteins. Statistical analyses were performed with Student's t-test. *P<0.05; ***P<0.0005; NS, not significant.

Fig 4: Osteogenic differentiation in response to Osteolectin requires ß-catenin.(A and B) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml recombinant mouse Osteolectin, as well as DMSO or 200 nM of the GSK3 inhibitor AZD2858. (A) Cells were lysed 24 hr later and immunoblotted for phospho-GSK3, ß-catenin, and Actin. (B) Alizarin red staining after 14 days (MC3T3-E1 cells) or 21 days (hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). (C and D) MC3T3-E1 cells, hBMSC#1 cells, and hBMSC#2 cells were transferred into osteogenic differentiation medium with PBS or 30 ng/ml Osteolectin, as well as DMSO or 200 nM of the ß-catenin inhibitor IWR-1-endo. (C) Cells were lysed 24 hr later and immunoblotted for phospho-GSK3, ß-catenin, and Actin. (D) Alizarin red staining after 14 days (MC3T3-E1 cells) or 21 days (hBMSC cells) to quantify osteoblast differentiation and mineralization (n = 3 independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Tukey’s multiple comparisons tests.10.7554/eLife.42274.008Figure 3—source data 1.Data for Data for Figure 3.

Fig 5: Oligomeric Aβ42 treatment induces GSK-3 activation and pathogenic phosphorylation of both APP and tau in primary rat cortical neurons. Primary rat cortical neurons were treated with 2.5 or 5 µm oligomeric Aβ42 for 24 h, and neuronal lysates were analyzed by Western blot using P-GSK3αβ (A) and total GSKαβ (B) antibodies. C, The bar graph represents the quantitative analysis of P-GSK3αβ levels normalized to total GSK3αβ expression. Membranes were then probed with P-Thr668 APP (D) and phospho-tau PHF-1 (F) antibodies. Finally, the membranes were reprobed with Actin antibody (H), which served as loading control. The bar graphs represent the quantitative analysis of P-Thr668 APP levels normalized to Actin (E) and PHF-1 levels normalized to Actin (G). Statistical analysis was performed using Student’s t test, and the data represent the mean ± SEM from three independent experiments (n = 3); *p < 0.05. I, Neurons were treated with indicated concentrations of Aβ and immunostained with neuron-specific MAP2 mouse monoclonal antibody and P-Thr668-APP rabbit polyclonal antibody. Immunostaining was visualized with Alexa Fluor 594 (red) and 488 (green) fluorophores, respectively. Hoechst staining was used to visualize nuclei (blue). The staining was analyzed with AxioVision Rel 4.8 software for Zeiss microscope. The corresponding bar graph (J) represents the qualitative analysis of the corrected total cell fluorescence for P-APP (n = 30 neurons/group). Statistical analysis was performed using ANOVA, and the data are representative of three independent experiments. Magnification 63×.