201123 n a mcherry rab11a s25n schuh  (Addgene inc)

Journal: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: A Primary hippocampal neurons (DIV6+4) were transfected with myc‐tagged TDP‐43 wild type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 μm. B Primary hippocampal neurons (DIV6+3) were transfected with either TDP‐43 wild type, TDP‐43ΔNLS, or an empty vector control together with GFP‐RAB11 to visualize recycling endosomes and analyzed as in Fig . C, D Quantitative analysis of recycling endosome movement (C) and vesicle number (D). Mean ± s.e.m., n = 4, one‐way ANOVA with Tukey's post‐test: * P < 0.05, ** P < 0.01.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Control, Immunostaining

Journal: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: A Primary hippocampal neurons (DIV6+3) were transfected with shCtrl and empty vector control or shTDP with either empty vector control, TDP‐43 wild type, or the ΔNLS variant together with GFP‐RAB11 to visualize recycling endosomes. Representative dendrite segments and kymographs of GFP‐RAB11 vesicle movement. Scale bar represents 12 s (vertical) and 25 μm (horizontal). B, C Quantitative analysis of vesicle motility (B) and the number (C) from kymographs in (A). Mean ± s.e.m., n = 5, one‐way ANOVA: * P < 0.05.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Transfection, Plasmid Preparation, Control, Variant Assay

Journal: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: A, B Luciferase assay to analyze transcriptional regulation of VPS4B by TDP‐43. (A) Reporter construct expressing Renilla luciferase (R‐Luc) driven by a VPS4B promoter fragment and firefly luciferase (FF) under the TK promoter. (B) HEK293 cells were transfected with shRNAs targeting human TDP‐43, a control shRNA, TDP‐43 wild type, or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter‐driven Renilla luciferase activity was normalized to TK promoter‐driven firefly luciferase. Quantification from six independent experiments. C, D ChIP assay to analyze binding of TDP‐43 to VPS4B promoter region in rat cortical neurons, HEK293 cells, and human brain tissue. PCR from input, negative control, and TDP‐43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percentage of input. E–H Analysis of VPS4B in the tissues of seven patients with the neuropathological diagnosis of FTLD/ALS‐TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 μm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed. Data information: Mean ± s.e.m., unpaired, two‐tailed t ‐test: * P < 0.05, ** P < 0.01. Source data are available online for this figure.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Luciferase, Construct, Expressing, Transfection, Control, shRNA, Plasmid Preparation, Activity Assay, Binding Assay, Negative Control, Biomarker Discovery, Immunofluorescence, Two Tailed Test

Journal: Cell Discovery

Article Title: Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

doi: 10.1038/celldisc.2016.17

Figure Lengend Snippet: RAB11A and IPO5 govern nuclear localization of PTAFR in hRMECs. ( a ) Co-immunoprecipitation (co-IP) of PTAFR with three major rabs controlling GPCR trafficking at indicated time-points following PAF C-16 stimulation. RAB5A (top row) co-IPs with only PTAFR following stimulation (early endocytosis ). RAB7A (second row) also co-IPs following stimulation and peaks at around 2 h (receptor targeted for degradation ). RAB11A (third row) co-IPs with PTAFR at all tested time-points, even in the absence of PAF C-16 stimulation (first column). The last row shows PTAFR as a loading control. ( b ) Knockdown of RAB11A using specific siRNA in hRMECs. The quantification of three westerns using the ImageJ software reveals ~75% reduction in nuclear immunoreactivity, as compared with that at PM. PM PTAFR is slightly affected (to much lesser extent), possibly due to recycling function of RAB11A. **** P <0.0001. ( c ) TEM on hRMECs transfected with either scrambled or specific RAB11A siRNAs. RAB11A knockdown specifically affects nuclear localization of PTAFR. Red arrows indicate nuclear labeling, whereas green arrows point labeling at PM. The TEM images are representative of three replicates. Scale bar=0.5 μm. ( d ) Overexpression of constitutively active (Q70L) or dominant-negative (S25N) RAB11A mutants in hRMECs (heterogeneous expression). The Q70L and S25N mutants resulted in ~125% and ~70% nuclear localization of PTAFR, respectively, as compared with non-transfected hRMECs with endogenous RAB11A levels (set at 100%) and normalized against LBR levels in all nuclear fractions. **** P <0.0001 and *** P <0.001 ( e ) siRNA-mediated knockdown of IPO5 in hRMECs. PM PTAFR is unaffected. The quantification of western blots reveals >90% reduction in nuclear immunoreactivity, as compared with that at PM. The values were normalized against LBR and cadherin in NUC and PM fractions, respectively. **** P <0.0001. ( f ) Co-transfection of HEK293T cells with PTAFR and IPO5. The overexpression of both proteins results in nuclear localization of PTAFR in HEK293T cells. All western blots are representative of three independent experiments. ( g ) Endogenous levels of IPO5 mRNA in hRMECs, CHO-K1 and HEK293T cells. HEK293T cells show negligible endogenous expression of IPO5.

Article Snippet: The RAB11A constructs GFP-rab11 DN (S25N) and EGFP-Rab11A-Q70L were gifts from Richard Pagano (Addgene; plasmid no. 12678) and Marci Scidmore (Addgene, Cambridge, MA, USA; plasmid no. 49553), respectively [ , ].

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Knockdown, Software, Transfection, Labeling, Over Expression, Dominant Negative Mutation, Expressing, Western Blot, Cotransfection

Journal: Cell Discovery

Article Title: Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

doi: 10.1038/celldisc.2016.17

Figure Lengend Snippet: Schematic diagram showing intracellular trafficking of PTAFR. Once synthesized in ER and glycosylated in TGN, Rab11a and Ipo5 together control (pathway highlighted by blue vesicles) nuclear localization of Ptafr, possibly directly from TGN. The nuclear PTAFR can be activated by the production of local PAF from membrane phospholipids by nuclear cPLA2 and ER-localized Lyso-PAF-acetyltransferase (Lyso-PAF-AT) . The nuclear Ptafr, in turn, activates the expression of proangiogenic genes such as Nos3 , Vegfa (indicated by solid line), whereas PM Ptafr regulated the expression of proinflammatory cytokines such as Il1b (indicated by dotted line). The role of N-glycosylation in the second extracellular domain of the receptor has been proposed in the cell surface targeting of Ptafr from TGN .

Article Snippet: The RAB11A constructs GFP-rab11 DN (S25N) and EGFP-Rab11A-Q70L were gifts from Richard Pagano (Addgene; plasmid no. 12678) and Marci Scidmore (Addgene, Cambridge, MA, USA; plasmid no. 49553), respectively [ , ].

Techniques: Synthesized, Control, Membrane, Expressing, Glycoproteomics