Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a,b) Western blot analysis of MeCP2 S421 phosphorylation and the quantification of total MeCP2 protein level in WT and phosphor-mutant aNPCs. (p=0.718, unpaired t-test with Welch’s correction, n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in WT aNPCs under normal proliferating condition and FGF2/EGF withdrawal for 24 hours. (n=3 in each group) (e) Western blot analysis reveals that MeCP2 S421 phosphorylation is induced by synchronizing aNPCs with nocodazole (150ng/ml). (f,g) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs under conditions: 1) DMSO, 2) 36 hours of nocodazole treatment, 3) 24 hours of roscovitine (25 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) Numbers next to Western blots are molecular weight markers. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Western Blot, Phospho-proteomics, Mutagenesis, Molecular Weight

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a,b) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in aNPCs: 1) DMSO, 2) 36 hours of nocodazole (150ng/ml) treatment, 3) 24 hours of hesperadin (2 μM) treatment after pre-synchronization of the cells by nocodazole for 12 hours. (n=3 in each group) (c,d) Western blot analysis and quantification of the relative MeCP2 S421 phosphorylation level in EGFP-shRNA or AURKB-shRNA lentivirus infected aNPCs, which are treated with DMSO or nocodazole for 24 hours. (e,f) Western blot analysis reveals that MeCP2 and AURKB are co-immunoprecipitated reciprocally. (g) Western blot analysis reveals endogenous interaction between MeCP2 and AURKB in aNPCs. (h) In vitro kinase assay followed by Western blot demonstrates that AURKB can phosphorylate S421 on MeCP2. Numbers next to Western blots are molecular weight markers.

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Western Blot, Phospho-proteomics, shRNA, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Molecular Weight

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a) Representative images of aNPCs isolated from WT and Mecp2 S421A;S424A/y hippocampus with BrdU pulse labeling, followed by immunocytochemistry analysis (b) Quantification of the percentage of BrdU/Sox2/Nestin triple-labeled cells in WT and Mecp2 S421A;S424A/y aNPCs. (n=3 in each group) (c) Representative images of Tuj1+ neurons differentiated from WT and Mecp2 S421A;S424A/y aNPCs (d) Quantification of the percentage of Tuj1+ cells in WT and Mecp2 S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (e) Representative images of GFAP+ astrocyte differentiated from WT and Mecp2 S421A;S424A/y aNPCs (f) Quantification of the percentage of GFAP+ cells in WT and Mecp2 S421A;S424A/y aNPCs upon differentiation. (n=3 in each group) (g) Relative mRNA level of neuronal marker ( Tuj1 and NeuroD1 ) and astrocyte marker ( GFAP ) in WT and Mecp2 S421A;S424A/y aNPCs upon differentiation, assayed by RT-qPCR. (n=3 in each group) (h) Schematics of the design of the in vivo BrdU labeling experiment. (i) Representative images of WT and the Mecp2 S421A;S424A/y brain sections stained for BrdU immunoreactivity. (j) Quantification of relative number of BrdU+ cells obtained through stereological counting from WT and Mecp2 S421A;S424A/y mice (n=9 in each group). (k) Quantification of the relative number of Ki67+ cells obtained through stereological counting from WT and Mecp2 S421A;S424A/y mice (n=6 in each group). (l) Schematics of the design of in vivo BrdU pulse/chase experiment to examine the differentiation profile of the adult-born hippocampal cells. (m) Representative confocal microscopy images to demonstrate how each cell type is identified. Three adult-born neurons (co-stained by BrdU and NeuN) are marked by arrow. One adult-born glial cell (co-stained by BrdU and S100b) is marked by arrowhead. Two adult-born undetermined cells (stained by BrdU only) are marked by asterisk. The rectangle panel to the right of the merged channel image is the y-z view of the same optical stack. The optical size of the z-scan is 0.4μm/step. (n) Quantification of proportions of the cell fate choices made by the dividing aNPCs in the hippocampus of WT and Mecp2 S421A;S424A/y mice. All scale bars are 50 μm. The bar graph shows the mean ± s.e.m * p<0.05 ** p<0.01

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Isolation, Labeling, Immunocytochemistry, Marker, Quantitative RT-PCR, In Vivo, Staining, Pulse Chase, Confocal Microscopy

Journal: Nature communications

Article Title: Cell cycle-linked MeCP2 phosphorylation modulates adult neurogenesis involving the Notch signaling pathway

doi: 10.1038/ncomms6601

Figure Lengend Snippet: (a) RT-qPCR analysis of the relative mRNA level of Dll1 and Notch1 in WT and Mecp2 S421A;S424A/y aNPCs. (n=5 in each group for Dll1 , n=8 in each group for Notch1 ) (b) RT-qPCR analysis of the relative mRNA level of Notch target gene Hes5 and Hey1 in WT and Mecp2 S421A;S424A/y aNPCs. (n=5 in each group) (c,d) Western blot analysis and quantification of the relative protein level of DLL1 in WT and Mecp2 S421A;S424A/y aNPCs. (n=4 in each group) (e, f) Western blot analysis and quantification of NICD level in WT and Mecp2 S421A;S424A/y aNPCs (n=5 in each group) (g) RT-qPCR analysis of the relative mRNA level of Hes5 and Hey1 in WT and Mecp2 S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (h, i) Representative images and quantification of BrdU-labeled cells in WT and Mecp2 S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus, followed by BrdU pulse labeling. (n=3 in each group) (j, k) Representative images and quantification of Tuj1+ neurons differentiated from WT and Mecp2 S421A;S424A/y aNPCs infected with GFP- or NICD-lentivirus. (n=3 in each group) (l) Relative mRNA level of neuronal marker ( Tuj1 and NeuroD1 ) and astrocyte marker ( GFAP ) in WT and Mecp2 S421A;S424A/y aNPCs, which are infected with GFP- or NICD-lentivirus and then cultured in differentiation condition. (n=3 in each group) Scale bar 50 μm. (m) ChIP-qPCR analysis of the promoter occupancy of WT and phosphor-mutant MeCP2 on Dll1 and Notch1 promoters. (n=4 in each group) Numbers next to Western blots are molecular weight markers. The bar graphs in this figure show the mean ± s.e.m * p<0.05 ** p<0.01

Article Snippet: 0.5 μg of Recombinant MeCP2 (Prospec, PRO-212) was incubated with AURKB-flag, AURKB-DN-flag or EGFP-flag, which were freshly immunopurified from 293T cells, in Kinase buffer (50mM HEPES, pH 7.5, 150mM NaCl, 10mM MgCl 2 , 1mM DTT, protease inhibitor (Roche), Phosphatase inhibitor (Sigma-Aldrich)) with or without 100μM ATP at 30 °C for 30 minutes.

Techniques: Quantitative RT-PCR, Western Blot, Infection, Labeling, Marker, Cell Culture, ChIP-qPCR, Mutagenesis, Molecular Weight