Journal: Frontiers in Cellular Neuroscience

Article Title: Identification of a Protein Network Driving Neuritogenesis of MGE-Derived GABAergic Interneurons

doi: 10.3389/fncel.2016.00289

Figure Lengend Snippet: Morphological maturation of MGE-derived interneurons . See also Figure . (A) Differentiation of MGE-dissociated cells from E14.5 mouse embryos plated on coverslips with PLL and laminin-1, and fixed at (DIV1), DIV3, or DIV6. Scale bar, 40 μm. (B–D) Maturation of MGE-derived GABAergic interneurons in vitro . (B) Increasing expression of neuronal and GABAergic markers detected by immunofluorescence. Scale bar, 40 μm. (C) Localization of tau-positive axons (arrowheads) and MAP2-positive dendrites (arrows) in DIV9 interneurons. The asterisk in the merge of the lower panel shows an example of a tau-positive axon running parallel and very close to a MAP2-positive dendrite. Scale bar, 40 μm. (D) Quantification of the percentage of cells positive for the indicated neuronal markers at DIV1–DIV6 ( n = 368 for calbindin and GAD67; n = 186 for GABA; n = 193 for TuJ1; n = 74 for MAP2 and tau; n = 71 for somatostatin, SST). (E) Left: by DIV15 most cells are positive for GABA. Center: immunostaining of DIV20 interneurons for VGAT and gephyrin. Right: 5-fold enlargement of the boxed region to show morphologically defined inhibitory synapses identified by the juxtaposition of VGAT-positive presynaptic terminals with gephyrin-positive postsynaptic terminals (arrows). Scale bars, 40 μm.

Article Snippet: Primary antibodies (Abs) used for immunofluorescence included: rabbit polyclonal Abs raised against αPIX (ARHGEF6 from Sigma), βPIX (si255 described previously; (Paris et al., ; Za et al., )), GABA (Sigma), GFP (Invitrogen), MAP2 (Santa Cruz), VGAT (Synaptic System), FLAG (Sigma), anti-PAK1 (crossreactive with PAK2 and PAK3, from Santa Cruz), liprin-α1 and anti-Rac3 (Proteintech), phospho-Src (Tyr416; Cell Signaling), phospho-Cofilin 1 (Serine 3; Santa Cruz), N-WASP (kindly provided by Rajat Rohatgi); goat pAb anti-βPAK/PAK3 (Santa Cruz); chicken pAb anti-GFP (Abcam); mAbs raised against Cre (mouse IgG 1 ; Covance), GAD67 (IgG 2a ; Millipore), gephyrin (IgG 1 ; Synaptic System), PKL/GIT (IgG 1 recognizing both GIT1 and GIT2; BD Bioscience), MAP2 (IgG 1 ; (Sigma), tau-1 (IgG 2a ; Millipore), TuJ1 (IgG 2a ; Covance), tubulin (Sigma), Rac1 (recognizing both Rac1 and Rac3, from BD Bioscience), HA (Primm Biotech), ERC1 (IgG 2a ELKS-30 from Abcam), FLAG (IgG 1 ; Sigma), Calbindin (from Swant), somatostatin (rat IgG2b; Millipore), cortactin (mouse IgG1; Millipore), VASP (9A2 rabbit mAb; Cell Signaling), cofilin (mouse mAb22 kindly provided by Hiroshi Abe, Bamburg laboratory), Src (mouse mAb; kindly provided by Margaret Jones, EMBL).

Techniques: Derivative Assay, In Vitro, Expressing, Immunofluorescence, Immunostaining

Journal: Frontiers in Cellular Neuroscience

Article Title: Identification of a Protein Network Driving Neuritogenesis of MGE-Derived GABAergic Interneurons

doi: 10.3389/fncel.2016.00289

Figure Lengend Snippet: Down-regulation of proteins of the PIX/GIT complex impairs dendritic development . See also Figures , . (A,C,E,H) MGE-derived interneurons transfected at DIV1 and cultured with BDNF until DIV6; immunostaining for GFP. (E) Neurons immunostained for the axonal marker tau (left) and GFP (right). Arrows show examples of axons costained for tau and GFP. Scale bars, 40 μm. (B,D,I,J) Effects of the expression of shRNAs on neurites (Sholl analysis). Bars, means ± SEM. In (B) , n = 86 neurons for shLuc, 29 neurons for shαPIX; 70 neurons for shβPIX.1, 33 neurons for shβPIX.2; in (D) n = 41 neurons for shLuc, 36 neurons for shGIT1, 22 neurons for shGIT2; in (I) n = 34 neurons for shLuc, 44 neurons for shPAK3; in (J) n = 68 neurons for shLuc, 41 neurons for shLip-α1, 43 neurons for shERC1. * p < 0.05; ** p < 0.01. (F–G) Effects of the indicated shRNAs on MAP2-positive dendrites (F) and tau-positive axons (G) . Means ± SEM; n = 10–11 neurons for shLuc, 12–13 neurons for shβPIX.1, 10–17 neurons for shGIT1; ** p < 0.01.

Article Snippet: Primary antibodies (Abs) used for immunofluorescence included: rabbit polyclonal Abs raised against αPIX (ARHGEF6 from Sigma), βPIX (si255 described previously; (Paris et al., ; Za et al., )), GABA (Sigma), GFP (Invitrogen), MAP2 (Santa Cruz), VGAT (Synaptic System), FLAG (Sigma), anti-PAK1 (crossreactive with PAK2 and PAK3, from Santa Cruz), liprin-α1 and anti-Rac3 (Proteintech), phospho-Src (Tyr416; Cell Signaling), phospho-Cofilin 1 (Serine 3; Santa Cruz), N-WASP (kindly provided by Rajat Rohatgi); goat pAb anti-βPAK/PAK3 (Santa Cruz); chicken pAb anti-GFP (Abcam); mAbs raised against Cre (mouse IgG 1 ; Covance), GAD67 (IgG 2a ; Millipore), gephyrin (IgG 1 ; Synaptic System), PKL/GIT (IgG 1 recognizing both GIT1 and GIT2; BD Bioscience), MAP2 (IgG 1 ; (Sigma), tau-1 (IgG 2a ; Millipore), TuJ1 (IgG 2a ; Covance), tubulin (Sigma), Rac1 (recognizing both Rac1 and Rac3, from BD Bioscience), HA (Primm Biotech), ERC1 (IgG 2a ELKS-30 from Abcam), FLAG (IgG 1 ; Sigma), Calbindin (from Swant), somatostatin (rat IgG2b; Millipore), cortactin (mouse IgG1; Millipore), VASP (9A2 rabbit mAb; Cell Signaling), cofilin (mouse mAb22 kindly provided by Hiroshi Abe, Bamburg laboratory), Src (mouse mAb; kindly provided by Margaret Jones, EMBL).

Techniques: Derivative Assay, Transfection, Cell Culture, Immunostaining, Marker, Expressing

Journal: Cell reports

Article Title: Huntingtin-mediated axonal transport requires arginine methylation by PRMT6

doi: 10.1016/j.celrep.2021.108980

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Chicken polyclonal anti-MAP2 , Merck Millipore , Cat#AB15452; RRID: AB_805385.

Techniques: Transduction, Virus, Control, Recombinant, Protease Inhibitor, Staining, shRNA, Plasmid Preparation, Software, Transfection