Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A ) Analysis in third instar larval fillet preparations expressing Ret-mCherry (anti-DsRed immunostaining) in class IV da neurons with ppk-Gal4 > CD8-GFP (anti-GFP). Ret was fairly evenly distributed in low and high order dendrites and the axon (yellow arrow). Scale bar 25 μm. ( B ) S2 cells were co-transfected with mys and Ret and imunostained with specific anti-mys (green) and anti-Ret antibodies (magenta). Mys and Ret colocalization in S2 cells is particularly strong in filopodial tips (arrows). Scale bar 1 μm. ( C ) S2 cells were transfected with V5-tagged Ret and flag-tagged mys with or without mew co-transfection. Mys-flag was immunoprecipitated and Ret interaction was probed by Western blotting with an anti-V5 antibody. The asterisks indicate secondary antibody cross-reactivity with the IP antibody. DOI: http://dx.doi.org/10.7554/eLife.05491.011

Article Snippet: Primary antibodies used were: rabbit anti-DsRed (1:500, BD Clontech), mouse anti-mew (1:100, DSHB), mouse anti-mys (1:100, DSHB).

Techniques: Expressing, Immunostaining, Transfection, Cotransfection, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

doi: 10.7554/eLife.05491

Figure Lengend Snippet: ( A ) Cell surface immunostaining of pHluorin-Ret-mCherry expressed in C4da neurons using ppk-Gal4 . Surface exposed Ret labeled by pHluorin (surface-stained with anti-GFP antibody) showed even labeling of the entire dendritic tree, while the mCherry signal displayed a more granular distribution of cellular Ret. Scale bar: 30 μm. ( B ) Cell surface immunostaining of pHluorin-Ret-mCherry co-expressing mys and mew in C4da neurons using ppk-Gal4 . Cell surface and cellular Ret were partially co-distributed with Mew in dendrites, as evident from fluorescence intensity plots along ( B′ ) low order and ( B′′ ) terminal dendrites. Scale bar 20 μm. ( C ) Quantitative colocalization analysis of Ret and integrins coexpressed in C4da neurons ( ppk-Gal4,CD8-GFP > Ret-mCherry,mys,mew ). Pearson coefficients were calculated for C4da neuron soma and dendrite regions showing a positive correlation of Ret and integrin signals (see ‘Materials and methods’ for details, mean ± SD, n = 5 per genotype). ( D and E ) Colocalization analysis of endogenous Ret and integrins overexpressed in C4da neurons ( ppk-Gal4,CD4-tdGFP > mys,mew ). Endogenous Ret signal was colocalized with ( D ) anti-mys or ( E ) anti-mew immunoreactivity in C4da neurons and colocalized pixels visualized in false color representations (coloc). ( D′ and E′ ) Stretches of terminal dendrites resliced in Z direction showing partial colocalization of ( D′ ) Ret and mys or ( E′ ) Ret and mew along the basal dendrite facing the ECM as indicated by arrows (see schematic drawing). Line intensity plots of the same dendrite portion show signal distribution of endogenous Ret and integrins together with the colocalized signals (coloc) and the CD4-tdGFP membrane marker. ( D′′ and E′′ ) Line intensity plots for a primary dendrite portion (indicated in D and E ). ( F ) Quantitative colocalization analysis of endogenous Ret and integrins expressed in C4da neurons ( ppk-Gal4,CD4-tdGFP > mys,mew ). Pearson coefficients calculated for C4da neuron soma and dendrites showing a positive correlation of endogenous Ret and integrin signals (see ‘Materials and methods’ for details, mean ± SD, n = 5 per genotype). DOI: http://dx.doi.org/10.7554/eLife.05491.010

Article Snippet: Primary antibodies used were: rabbit anti-DsRed (1:500, BD Clontech), mouse anti-mew (1:100, DSHB), mouse anti-mys (1:100, DSHB).

Techniques: Immunostaining, Labeling, Staining, Expressing, Fluorescence, Marker