Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: 15 kDa granulysin causes differentiation of monocytes to dendritic cells but lacks cytotoxic activity

doi: 10.4049/jimmunol.1200570

Figure Lengend Snippet: Granulysin is expressed by a subset of PBMCs. A, PBMCs from normal donors were stained with antibodies specific for either CD4 or CD8 plus CD27, CD45RA and granulysin. After gating on either CD4 or CD8, expression of CD27 and CD45RA was used to divide cells into memory (CD27+CD45RA−), naïve (CD27+CD45RA+) or effector (CD27−CD45RA+) cells. The percent of cells in each subset that stain for granulysin is shown. B, PBMCs were stained with antibodies specific for CD3, CD56 and granulysin. C, 107 CD8+ T cells or 106 NK cells were cultured for 2 days in medium with or without recombinant IL-15 (50 ng/ml). Where indicated, cells were activated with PMA plus ionomycin (P/I) for the final 6 hours of culture. Supernatants and cell pellets were subjected to SDS-PAGE and Western blot was carried out using anti-granulysin antibody. Results are representative of 10 donors (A,B) or 3 donors (C).

Article Snippet: Single cells suspensions of PBMCs were stained with fluorochrome-conjugated Abs specific for CD3, CD4, CD8, CD56, CD27, and CD45RA (BD Biosciences, San Jose, CA), fixed and permeabilized (BD Cytofix/Cytoperm, BD Biosciences), and then stained with rabbit anti-granulysin antiserum or preimmune rabbit serum ( 5 , 18 ).

Techniques: Staining, Expressing, Cell Culture, Recombinant, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein-tyrosine Phosphatase (PTP) Wedge Domain Peptides

doi: 10.1074/jbc.m603131200

Figure Lengend Snippet: FIGURE 7. LAR associates with TrkA and regu- lates its activation. A, PC12 cell extract coimmu- noprecipitates (IP) formed using preimmune serum (PreI), anti-TrkA antibody, or anti-LAR anti- body (directed against the LAR C terminus) were assessed by Western blot (W) using LAR C termi- nus antibody. LAR is captured in anti-TrkA and anti-LAR antibody immunoprecipitates. B, immu- noprecipitates formed using preimmune anti- body or anti-LAR antibody (directed against the LAR C terminus) were assessed by Western blot using anti-TrkA antibody followed by re-probing with anti-LAR antibody. TrkA and LAR are cap- tured in anti-LAR antibody immunoprecipitates. Exposure of PC12 cells for 10 min to either CM or NGF (0.18 nM) did not affect TrkA/LAR association. The addition of WLAR-Tat (WL-Tat), but not SLAR- Tat (SL-Tat), inhibited TrkA association with LAR. C, Western blot analysis for phospho-TrkA Tyr-490 (upper panel) and total TrkA protein (lower panel) in extracts derived from control null-transfected PC12 cells (LC-2) and in LAR antisense-transfected LAR-deficient PC12 cells (LAS-1) in the absence of NGF (CM) and after NGF (1.80 nM) treatment for the indicated durations. D, quantitation of West- ern blot signal demonstrates augmented NGF-in- duced activation of TrkA in LAR-deficient cells at the 10 min time point (***, p 0.001; n 5 assays; Student’s t test). E, Western blot analysis for phos- pho-TrkA (upper panel) and total TrkA protein (lower panel) in PC12 cells treated with CM, NGF (0.18 nM), SLAR-Tat (4 M), or WLAR-Tat (4 M). F, quantitation of Western blot signal demonstrates the expected TrkA activation by NGF and also sig- nificant TrkA activation by WLAR-Tat compared with SLAR-Tat control (**, p 0.001; n 6 assays). G, Western blot analysis for phospho-TrkA (upper panel) and total TrkA (lower panel) in PC12 cells treated with CM, NGF (0.18 nM), NGF (0.18 nM) SLAR-Tat (4 M), or NGF (0.18 nM) WLAR-Tat (4 M). H, quantitation of Western blot signal dem- onstrates the expected TrkA activation by NGF and a significant augmentation of TrkA activation by WLAR-Tat compared with SLAR-Tat control (***, p 0.001; n 6 assays).

Article Snippet: Immunoprecipitations were performed by adding preimmune serum (5 g), polyclonal anti-TrkA antibody (Cell Signaling, Beverly,MA; 1:250 dilution), or polyclonal anti-LAR antibody directed against the LARC terminus (5 g) and incubating overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Derivative Assay, Control, Transfection, Quantitation Assay

Journal: Acta Neuropathologica Communications

Article Title: Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

doi: 10.1186/s40478-020-00956-z

Figure Lengend Snippet: Confocal microscopy and ultrastructural imaging of early endosomes in fibroblasts from individuals with DS. a Representative images of EEA1-positive early endosomes (green) in fibroblasts from euploid individuals and individuals with DS; b Quantification of EEA1-positive puncta volume from confocal microscopy images of fibroblasts, showing a significantly increased volume of early endosome in DS condition (mixed effect ANOVA, genotype * p -value = 0.03); c EEA1-positive puncta number normalized to ROI area is not significantly different between euploid and DS fibroblasts in confocal microscopy (mixed effect ANOVA, ns, genotype p -value = 0.73); d - g Electron micrographs of EEA1 immunoperoxidase-labelled fibroblasts from a euploid individual ( d squared zone magnified in ( e )) and an individual with DS ( f squared zone magnified in ( g )) after chemical fixation

Article Snippet: Free floating brain sections were washed three times in PBS and pre-incubated in blocking and permeabilization solution (normal donkey serum 5% / Triton X-100 0.2% in PBS) for 1 h. Brain sections were incubated overnight in primary antibody solution: goat anti-choline-acetyltransferase (ChAT) antibody (polyclonal, AB144P, Millipore, 1/300, kindly provided by Dr. Sylvie Berrard, Hôpital universitaire Robert-Debré, Paris, France), mouse anti-NeuN antibody (monoclonal, MAB377, Millipore, 1/500) and rabbit anti-EEA1 antibody (C45B10, Cell Signaling, 1/400).

Techniques: Confocal Microscopy, Imaging

Journal: Acta Neuropathologica Communications

Article Title: Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

doi: 10.1186/s40478-020-00956-z

Figure Lengend Snippet: Ultrastructural imaging of early endosomes in LCLs from individual with DS. a , b Electron micrograph representing EEA1 immunoperoxidase-labelled early endosomes in a LCL from a euploid individual. The squared zone in ( a ) is magnified in ( b ); c , d 2 serial TEM sections of an EEA1 immunoperoxidase-labelled cluster of early endosomes in a trisomic lymphoblastoid cell; e , f 2 clusters of early endosomes in a lymphoblastoid cell from an individual with DS. The pseudo-colored red halo suggests the extent of labelled area as it should appear with confocal analysis. The squared zone in ( c ) is enlarged in ( d ); g Quantification of early endosomal area in LCLs imaged by TEM, showing no significant difference in mean early endosomal area between LCLs from euploid individual and individual with DS (mixed effects ANOVA, genotype, p -value = 0.57)

Article Snippet: Free floating brain sections were washed three times in PBS and pre-incubated in blocking and permeabilization solution (normal donkey serum 5% / Triton X-100 0.2% in PBS) for 1 h. Brain sections were incubated overnight in primary antibody solution: goat anti-choline-acetyltransferase (ChAT) antibody (polyclonal, AB144P, Millipore, 1/300, kindly provided by Dr. Sylvie Berrard, Hôpital universitaire Robert-Debré, Paris, France), mouse anti-NeuN antibody (monoclonal, MAB377, Millipore, 1/500) and rabbit anti-EEA1 antibody (C45B10, Cell Signaling, 1/400).

Techniques: Imaging

Journal: Acta Neuropathologica Communications

Article Title: Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

doi: 10.1186/s40478-020-00956-z

Figure Lengend Snippet: Confocal microscopy and TEM imaging of BFCNs from 4-month-old Ts65Dn mice. a Representative confocal images of DAPI (blue), ChAT (red), EEA1 (green) labelling and overlay in BFCNs from WT and Ts65Dn mice. For clarity, NeuN staining is not shown; b Quantification of EEA1-positive puncta surface at equatorial section of BFCNs shows a significant increase in mean area in Ts65Dn mice as compared to WT mice (mixed effects ANOVA, genotype * p -value = 0.04); c Low magnification micrograph of a ChAT gold-labelled BFCNs of a Ts65Dn mouse. Early endosomes are labelled by an EEA1-DAB precipitate. The soma profile is pseudo-colored in yellow for clarity. N indicates the nucleus and asterisks show two lamellar bodies, an organelle specifically found in cholinergic neurons. The squared zone is enlarged in ( d ) showing an isolated DAB-EEA1-labelled endosome and a cluster of at least 2 endosomes, the latter being magnified in ( e ). The pseudo-colored red halo in ( e ) suggests the extent of labelled area as it should appear with confocal analysis

Article Snippet: Free floating brain sections were washed three times in PBS and pre-incubated in blocking and permeabilization solution (normal donkey serum 5% / Triton X-100 0.2% in PBS) for 1 h. Brain sections were incubated overnight in primary antibody solution: goat anti-choline-acetyltransferase (ChAT) antibody (polyclonal, AB144P, Millipore, 1/300, kindly provided by Dr. Sylvie Berrard, Hôpital universitaire Robert-Debré, Paris, France), mouse anti-NeuN antibody (monoclonal, MAB377, Millipore, 1/500) and rabbit anti-EEA1 antibody (C45B10, Cell Signaling, 1/400).

Techniques: Confocal Microscopy, Imaging, Staining, Isolation

Journal: Acta Neuropathologica Communications

Article Title: Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

doi: 10.1186/s40478-020-00956-z

Figure Lengend Snippet: Confocal microscopy and SR-SIM of human isogenic iPSCs-derived neurons from an individual with T21 mosaicism. a Confocal z-projected representative images of DAPI (blue), MAP 2 (red), EEA1 (green) labelling in euploid and isogenic T21 iPSCs-derived neurons; b Quantification of the mean EEA1-positive puncta volume in confocal microscopy images reveals significantly increased volume in T21 neurons as compared to euploid isogenic neurons (mixed effects ANOVA, genotype * p -value = 0.03); c Quantification of EEA1-positive puncta number normalized to the ROI area shows a significant increase (Mann-Whitney test, ** p -value = 0.0043); d Representative z-projected images of a T21 neuron imaged by confocal microscopy (left) and SR-SIM (right). A cluster of EEA1-positive puncta observed by SR-SIM appearing as enlarged puncta by confocal microscopy is magnified (scale bar = 500 nm); e Quantification of EEA1-positive puncta volume in SR-SIM images shows no significant difference (mixed effects ANOVA, genotype, p -value = 0.95); f The mean number of EEA1-positive puncta clusters per cell is significantly increased in T21 neurons as compared to euploid isogenic neurons (Mann-Whitney test, * p -value = 0.047)

Article Snippet: Free floating brain sections were washed three times in PBS and pre-incubated in blocking and permeabilization solution (normal donkey serum 5% / Triton X-100 0.2% in PBS) for 1 h. Brain sections were incubated overnight in primary antibody solution: goat anti-choline-acetyltransferase (ChAT) antibody (polyclonal, AB144P, Millipore, 1/300, kindly provided by Dr. Sylvie Berrard, Hôpital universitaire Robert-Debré, Paris, France), mouse anti-NeuN antibody (monoclonal, MAB377, Millipore, 1/500) and rabbit anti-EEA1 antibody (C45B10, Cell Signaling, 1/400).

Techniques: Confocal Microscopy, Derivative Assay, MANN-WHITNEY