Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours. Under these conditions EHD1 was seen to co-localize with SMO in the primary cilia. A profile scan for SMO and EHD1 in the primary cilia merged panel is shown. Under these conditions, EHD1 was seen to traffic to the cilia of 60% cells studied. ( B ) WT NIH3T3 cells stably expressing SMO-GFP and transiently expressing EHD1-DsRed were starved in low serum media for 24 hours and stimulated with SAG immediately before starting live imaging of Smoothened and EHD1.As reported in earlier studies, Smoothened was found in preciliary vesicles under non-stimulated conditions but upon SHH pathway activations, EHD1 was seen to associate with Smoothened vesicles and co-traffic with SMO into the primary cilia.

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Stable Transfection, Expressing, Imaging

Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) Immunoblotting was used to assess the presence of SMO in anti-EHD1 immunoprecipitates from WT NIH3T3 cells stably expressing SMO GFP treated with SAG (24 hrs). Input samples show the amount of each protein in the whole extract before the IP. Immunoblots showing the amount of SMO that co-precipitated with EHD1 from cells that stably expressed the protein and were treated with SAG for 24 hrs. A control IP was performed with non-specific rabbit IgG instead of EHD1 antibody and WT NIH3T3 cell lysates stably expressing SMO GFP treated with SAG (24 hrs). Arrows denote the molecular weight fraction of SMO that associates with EHD1. Full-length blots/gels are presented in . ( B ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours and lysed and these lysates were incubated with GST-EHD1 or GST-EH domain deleted EHD1. The membranes were probed with antibodies to Smoothened and GST.GST-fused to the empty plasmid vector was used as a negative control. The data is representative of three separate experimental repeats. Full-length blots/gels are presented in .

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Western Blot, Stable Transfection, Expressing, Control, Molecular Weight, Incubation, Plasmid Preparation, Negative Control