Journal: Frontiers in Cellular Neuroscience

Article Title: A selective histone deacetylase-6 inhibitor improves BDNF trafficking in hippocampal neurons from Mecp2 knockout mice: implications for Rett syndrome

doi: 10.3389/fncel.2014.00068

Figure Lengend Snippet: Activity-dependent release of BDNF-YFP is impaired in Mecp2 knockout neurons . (A) Representative examples of BDNF release in wildtype and Mecp2 knockout neurons: no treatment, stimulated with KCl, stimulated with KCl in the absence of extracellular Ca 2+ and stimulated with KCl upon TBA treatment; scale bar = 10 μm. BDNF-YFP is shown in green, while YFP immunoreactivity is shown in red. Yellow pixels represent colocalization of green and red pixels, i.e., secreted BDNF. (B) Proportion of released BDNF per total BDNF upon KCl stimulation in wildtype neurons and Mecp2 knockout neurons in the absence or presence of extracellular Ca 2+ , nifedipine (50 μ M), CNQX (10μ M) /D-APV (50 μ M), Bicuculline (10 μ M), TTX (0.5 μ M) or TBA (1 μ M). The proportion of released BDNF per total BDNF was normalized to that in non-stimulated neurons (Ctl), and expressed as % of Ctl. ** p < 0.01; wildtype vs. Mecp2 knockout; *** p < 0.001 compared to Mecp2 knockout Ctl group stimulated with KCl; # p < 0.05; ## p < 0.01 compared to wildtype Ctl group stimulated with KCl.

Article Snippet: Quantification of co-localized pixels was performed using Colocalization module in Imaris.

Techniques: Activity Assay, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

doi: 10.3390/ijms19071837

Figure Lengend Snippet: Panx2 and N86Q colocalize with markers of the endoplasmic reticulum and Golgi. Representative confocal micrographs of Panx2 and N86Q ectopically expressed in AD293 cells. Co-immunolabeling with anti-Panx2 antibody (green) and organelle markers (magenta): ( A ) PDI, endoplasmic reticulum (ER); ( B ) GM-130, cis-Golgi matrix. Panx2 has a perinuclear localization and is spread intracellularly in the cytoplasm partially colocalizing with markers of the endoplasmic reticulum and Golgi. N86Q aggregates also overlap with ER and Golgi markers and disrupt their distribution. Yellow arrowheads indicate representative regions of colocalization. Nuclei (blue) were counterstained with Hoechst 33342. Scale bars = 20 μm. Pearson Correlation Coefficients (right) were calculated for multiple regions of interest (ROI)s corresponding to double-labeled cells. Statistical significance was considered when p < 0.05 (*** p ≤ 0.0001, N = 3 independent experiments), using unpaired two-tailed t test with Welch’s correction. Error bars denote mean ± S.E.M.

Article Snippet: Colocalization was quantitated with the colocalization plug-in of the Zeiss software (ZEN, version 2.3, blue edition).

Techniques: Immunolabeling, Labeling, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

doi: 10.3390/ijms19071837

Figure Lengend Snippet: Panx2 and N86Q do not localize to late endosomes/lysosomes or mitochondria. Confocal micrographs of Panx2 and N86Q ectopically expressed in AD293 cells. Co-immunolabeling with anti-Panx2 antibody (green) and organelle markers (magenta): ( A ) Lamp-2, lysosomes and late endosomes, and ( B ) Mitotracker ® Red, mitochondria showed that neither Panx2 or N86Q mutant exhibited significant overlap with the markers. Nuclei (blue) were counterstained with Hoechst 33342. Nuclei (blue) were counterstained with Hoechst 33342. Scale bars = 20 μm. Pearson Correlation Coefficients (right) were calculated for multiples ROIs corresponding to double-labeled cells. There was no statistical significance ( p > 0.05, N = 3, unpaired two-tailed t test with Welch’s correction) in the degree of colocalization between Panx2 and N86Q with the markers. Error bars denote mean ± S.E.M.

Article Snippet: Colocalization was quantitated with the colocalization plug-in of the Zeiss software (ZEN, version 2.3, blue edition).

Techniques: Immunolabeling, Mutagenesis, Labeling, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

doi: 10.3390/ijms19071837

Figure Lengend Snippet: N-glycosylation may enhance plasma membrane localization of Panx2 when co-expressed with Panx1. Confocal micrographs of ( A ) Panx2-FLAG or ( B ) N86Q-FLAG (magenta) ectopically co-expressed with mouse Panx1 (green) in HEK293T cells. 72 h post-transfection Panx2-FLAG partially colocalized with Panx1 at the cell membrane (see black arrows in Linescan, panel A ) with a subpopulation still in intracellular compartments. N86Q-FLAG formed intracellular aggregates and showed limited colocalization with Panx1 at the plasma membrane (see black arrows in Linescan, panel B ). Yellow arrowheads denote regions of colocalization of Panx1 and FLAG labeling also depicted with black arrows in the corresponding linescans. Insets: Linescans showing the overlapping (black arrows) between fluorescence peaks to denote colocalization. Nuclei (blue, Hoechst 33342). Scale bars = 20 µm.

Article Snippet: Colocalization was quantitated with the colocalization plug-in of the Zeiss software (ZEN, version 2.3, blue edition).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Transfection, Labeling, Fluorescence