Journal: BioMed Research International

Article Title: Valproic Acid Protects Primary Dopamine Neurons from MPP + -Induced Neurotoxicity: Involvement of GSK3 β Phosphorylation by Akt and ERK through the Mitochondrial Intrinsic Apoptotic Pathway

doi: 10.1155/2017/8124501

Figure Lengend Snippet: VPA upregulated Akt and ERK1/2 activation and p-GSK3 β expression after MPP + -treatment, whereas inhibitors of PI3K and MAPK pathways counteracted VPA-induced GSK3 β phosphorylation in DA neurons. VPA (0.6 mM) was given in DA neuron cultures after treatment with 10 µ M MPP + for 48 h at DIV4. (a) Western blot analysis of p-Akt, Akt, p-ERK1/2, and ERK1/2 expression. (b) Quantification of Western blot data for relative p-Akt and (c) p-ERK activity. (d) Western blot analysis of GSK3 β and p-GSK3 β expression with or w/o PI3K and MAPK pathway inhibitors LY294002 (LY) and PD98059 (PD), respectively. (e) Quantification of Western blot data in (d) for relative p-GSK3 β activity. The data are from of six independent experiments. ∗ P < 0.05.

Article Snippet: Membranes were blocked with 5% nonfat milk solution in Tris -buffered saline with 0.1% Triton X-100 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with the primary antibody dilutions in TBST: Akt (1 : 1000), phospho-Akt (1 : 800), ERK1/2 (1 : 1000), phospho-ERK1/2 (1 : 800), GSK3 β (1 : 1000), and phospho-GSK3 β (pSer 9 ) (1 : 800; all from Cell Signaling Technology Inc.), Bax (1 : 800), Bcl-2 (1 : 1000), cytochrome c (1 : 1000), caspase-3 (1 : 800), and caspase-9 (1 : 800; all from Cell Signaling Technology Inc.).

Techniques: Activation Assay, Expressing, Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Tau Exon 10 Inclusion by PrP C through Downregulating GSK3β Activity

doi: 10.3390/ijms22105370

Figure Lengend Snippet: GSK3β activity in mice devoid of PrP C expression. ( A , B ) GSK3β activation analyzed by WB in brain extract from WT and Prnp 0/0 mice ZH1 or ZH3 at the age of 3 months. ( A ) Representative WB analysis using anti-phospho-tyr 279/216 GSK3 antibody (monoclonal 5G-2F) in parallel with anti-phospho-ser 9 GSK3 antibody (monoclonal 2D3) in each case. Membranes were re-probed with antibody against total GSK3 (monoclonal 4G-1E) for protein standardization. ( B ) Histograms showing the quantified ratio between phospho-tyr 279/216 and phospho-ser 9 after densitometry analysis of both phosphorylated GSK3β epitopes in each genotype, which represents the kinase activity. n = 3 mice were examined in each group and data represent the mean ± S.E.M. Differences between groups were considered statistically significant at ** p < 0.01 ( t -test).

Article Snippet: GSK3 (clone 4G-1E, WB, 1:3000), GSK3 phospho-tyr 279/216 (clone 5G-2F, WB, 1:2000) and GSK3 phospho-ser 9 (clone 2D3, WB, 1:250) were from Upstate Biotechnology, Lake Placid, NY, USA.

Techniques: Activity Assay, Expressing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Tau Exon 10 Inclusion by PrP C through Downregulating GSK3β Activity

doi: 10.3390/ijms22105370

Figure Lengend Snippet: Effects on tau, alternative exon 10 forms, and GSK3β in tau transgenic mice overexpressing tau-GFP after ablation of PrP C expression. ( A – C ) Total tau expression analyzed in brain extract from tau-GFP, tau-GFP- Prnp +/0 , and tau-GFP- Prnp 0/0 mice at the age of 3 months. ( A ) Representative WB analysis using total anti-tau antibody (monoclonal Tau5) in parallel with anti-PrP C antibody (monoclonal 6H4) in each case. Actin detection was used as control loading protein. ( B ) Histograms showing the densitometry study of tau-GFP expression in each genotype. ( C ) Histograms showing the densitometry study of endogenous tau expression in each genotype. ( D – G ) Endogenous 3R and 4R tau isoforms expression analyzed in brain extract from tau-GFP, tau-GFP- Prnp +/0 , and tau-GFP- Prnp 0/0 mice at the age of 3 months. ( D ) Representative WB analysis using anti-3R tau antibody (monoclonal RD3) in parallel with anti-4R tau antibody (monoclonal RD4) in each genotype. Actin detection was used as control loading protein. ( E , F ) Histograms showing the densitometry study of 3R tau ( E ) and 4R tau ( F ) expression in each genotype. ( G ) Graphical representation of the 3R/4R tau ratio analyzed with data represented in ( E , F ). ( H , I ) GSK3β activation analyzed with WB in brain extract from tau-GFP, tau-GFP- Prnp +/0 , and tau-GFP- Prnp 0/0 mice at the age of 3 months. ( H ) Representative WB analysis using anti-phospho-tyr 279/216 GSK3 antibody (monoclonal 5G-2F) in parallel with anti-phospho-ser 9 GSK3 antibody (monoclonal 2D3) in each case. Membranes were re-probed with antibody against total GSK3 (monoclonal 4G-1E) for protein standardization. ( I ) Histograms showing the quantified ratio between phospho-tyr 279/216 and phospho-ser 9 after densitometry analysis of both phosphorylated GSK3β epitopes in each genotype, which represents the kinase activity. n = 3 mice were examined in each group and data represents the mean ± S.E.M. Differences between groups were considered statistically significant at ** p < 0.01 and * p < 0.05 ( t -test).

Article Snippet: GSK3 (clone 4G-1E, WB, 1:3000), GSK3 phospho-tyr 279/216 (clone 5G-2F, WB, 1:2000) and GSK3 phospho-ser 9 (clone 2D3, WB, 1:250) were from Upstate Biotechnology, Lake Placid, NY, USA.

Techniques: Transgenic Assay, Expressing, Activation Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Tau Exon 10 Inclusion by PrP C through Downregulating GSK3β Activity

doi: 10.3390/ijms22105370

Figure Lengend Snippet: Effects on tau, alternative exon 10 forms, and GSK3β in tau transgenic mice overexpressing human P301S MAPT mutation after ablation of PrP C expression. ( A – C ) Total tau expression analyzed in brain extract from P301S and P301S- Prnp 0/0 mice at the age of 3 months. ( A ) Representative WB analysis using total anti-tau antibody (monoclonal Tau5) in parallel with anti-PrP C antibody (monoclonal 6H4) in each case. Actin detection was used as control loading protein. ( B ) Histograms showing the densitometry study of both endogenous and overexpressed tau expression in each genotype. ( C ) Immunohistochemical detection of total tau in mouse brain sections from P301S and P301S- Prnp 0/0 . Monoclonal tau5 antibody was used to detect increased immunoreaction in DG from P301S in contrast to P301S- Prnp 0/0 animals. Scale bars = 100 µm and 50 µm. ( D – G ) Endogenous 3R and 4R tau isoform expression analyzed in brain extract from P301S and P301S- Prnp 0/0 mice at the age of 3 months. ( D ) Representative WB analysis using anti-3R tau antibody (monoclonal RD3) in parallel with anti-4R tau antibody (monoclonal RD4) in each genotype. Actin detection was used as control loading protein. ( E , F ) Histograms showing the densitometry study of 3R tau ( E ) and 4R tau ( F ) expression in each genotype. ( G ) Graphical representation of the 3R/4R tau ratio analyzed with data represented in ( E , F ). ( H , I ) GSK3β activation analyzed with WB in brain extract from P301S and P301S- Prnp 0/0 mice at the age of 3 months. ( H ) Representative WB analysis using anti-phospho-tyr 279/216 GSK3 antibody (monoclonal 5G-2F) in parallel with anti-phospho-ser 9 GSK3 antibody (monoclonal 2D3) in each case. Membranes were re-probed with antibody against total GSK3 (monoclonal 4G-1E) for protein standardization. ( I ) Histograms showing the quantified ratio between phospho-tyr 279/216 and phospho-ser 9 after densitometry analysis of both phosphorylated GSK3β epitopes in each genotype, which represents the kinase activity. n = 3 mice were examined in each group and data represents the mean ± S.E.M. Differences between groups were considered statistically significant at * p < 0.05 ( t -test).

Article Snippet: GSK3 (clone 4G-1E, WB, 1:3000), GSK3 phospho-tyr 279/216 (clone 5G-2F, WB, 1:2000) and GSK3 phospho-ser 9 (clone 2D3, WB, 1:250) were from Upstate Biotechnology, Lake Placid, NY, USA.

Techniques: Transgenic Assay, Mutagenesis, Expressing, Immunohistochemical staining, Activation Assay, Activity Assay