Rade velocities (m.s regular deviation (SD)) of GFPbuy DFMTI tagged IFT proteins along amphid and phasmid channel cilia (combined; major rows), or phasmid cilia only (bottom rows). ttest pairwise comparison with wildtype controls, n quantity of particles, N measured number of amphids and phasmids. OSM may be the worm orthologue of KIF; CHE would be the worm orthologue of IFT; OSM would be the worm orthologue of IFT. b Representative fluorescence pictures of phasmid cilia displaying regular IFT protein localisations and distributions in tm mutants. ds distal segment, ms middle segment, bb basal body area, den dendrite. All MCB-613 web photos are similarly scaled and orientated (arrow denotes basal body). Scale bar, m. c Representative kymographs (time (t) more than distance (d) plots) applied to produce IFT price measurements. For every single kymograph, the horizontal axis (distance) is m along with the vertical axis (time) is seconds. d Distribution plots of IFT protein velocities. (JPG kb) Added file Data supplementary to the nocodazole destabilization assay shown in Fig a, b Replicate pictures of DMSO or nocodazoletreated hTERTRPE cells. Cells were transfected with SFTAPtagged KIAA (detected with antiFLAG immunostaining; green) or GFPKIAA and counterstained with antiacetylated tubulin (red) and DAPI (blue). Cells with high KIAA expression are characterised by a filamentous staining pattern and spots of accumulated KIAA signal. In nontransfected cells, minute nocodazole therapy resulted in the loss of a stabilised MT network (see specifically the high exposure pictures), as judged by loss of (pretty much) all cytoplasmic acetylated tubulin staining andor the absence of a filamentous staining pattern. In transfected cells (expressing KIAA), a filamentous acetylated tubulin staining pattern remained. See also Fig. for examples. Scale bar, m. c Quantification on the presence of a detectable filamentous acetylated alpha tubulin MT network in GFPKIAA transfected and nontransfected cells, treated with DMSO or M nocodazole for minutes. MT networks could possibly be identified in roughly of GFPKIAA transfected cells compared with untransfected cells (n). Resulting from the decrease expression level and transfection efficiency of GFPKIAA (compared with SFTAPtagged KIAA in Fig. c), only a fairly modest number of transfected cells might be analysed. (JPG kb) Additional file Outcomes in the SFTAP evaluation with overexpressed Nterminally SFTAPtagged KIAA in HEKT cells. Shown is the number of distinctive identified peptides too as the sequence coverage for every single protein detected by mass spectrometry. Proteins identified in out SFTAP control experiments (empty vector) had been removed. (XLSX kb) Additional file Supplementary details to the data in Fig a Schematic representation of each of the distinctive KIAA fragments made use of to screen our collection of ciliary proteins. The predicted protein repeat domains, shown in More files and , are depicted as d to d. Constructs had been generated containing isolated domains too as a mixture of domains. b Single transfections of PalMyrKIAA and mRFPKATNBL, showing PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17174591 that membrane localisation in the mRFP tagged protein is indeed dependent around the interaction together with the PalMyrtagged protein. (JPG kb) More file Postembryonic tissue expression of C. elegans katanin genes mei, mei and FG Shown are fluorescence photos of worms expressing a transcriptional GFP reporter under the c
ontrol on the indicated gene’s promoter, which stains the whole cell in which it is expressed. DiI (red) costain ident.Rade velocities (m.s common deviation (SD)) of GFPtagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). ttest pairwise comparison with wildtype controls, n quantity of particles, N measured number of amphids and phasmids. OSM may be the worm orthologue of KIF; CHE is definitely the worm orthologue of IFT; OSM could be the worm orthologue of IFT. b Representative fluorescence images of phasmid cilia showing standard IFT protein localisations and distributions in tm mutants. ds distal segment, ms middle segment, bb basal body region, den dendrite. All pictures are similarly scaled and orientated (arrow denotes basal body). Scale bar, m. c Representative kymographs (time (t) over distance (d) plots) utilized to generate IFT rate measurements. For every kymograph, the horizontal axis (distance) is m along with the vertical axis (time) is seconds. d Distribution plots of IFT protein velocities. (JPG kb) More file Information supplementary towards the nocodazole destabilization assay shown in Fig a, b Replicate photos of DMSO or nocodazoletreated hTERTRPE cells. Cells have been transfected with SFTAPtagged KIAA (detected with antiFLAG immunostaining; green) or GFPKIAA and counterstained with antiacetylated tubulin (red) and DAPI (blue). Cells with high KIAA expression are characterised by a filamentous staining pattern and spots of accumulated KIAA signal. In nontransfected cells, minute nocodazole treatment resulted in the loss of a stabilised MT network (see especially the higher exposure photos), as judged by loss of (just about) all cytoplasmic acetylated tubulin staining andor the absence of a filamentous staining pattern. In transfected cells (expressing KIAA), a filamentous acetylated tubulin staining pattern remained. See also Fig. for examples. Scale bar, m. c Quantification in the presence of a detectable filamentous acetylated alpha tubulin MT network in GFPKIAA transfected and nontransfected cells, treated with DMSO or M nocodazole for minutes. MT networks could be identified in approximately of GFPKIAA transfected cells compared with untransfected cells (n). As a result of the lower expression level and transfection efficiency of GFPKIAA (compared with SFTAPtagged KIAA in Fig. c), only a relatively tiny number of transfected cells may very well be analysed. (JPG kb) Extra file Final results with the SFTAP analysis with overexpressed Nterminally SFTAPtagged KIAA in HEKT cells. Shown may be the variety of distinctive identified peptides too as the sequence coverage for each and every protein detected by mass spectrometry. Proteins identified in out SFTAP control experiments (empty vector) had been removed. (XLSX kb) Additional file Supplementary info to the data in Fig a Schematic representation of all the diverse KIAA fragments utilized to screen our choice of ciliary proteins. The predicted protein repeat domains, shown in Further files and , are depicted as d to d. Constructs had been generated containing isolated domains too as a combination of domains. b Single transfections of PalMyrKIAA and mRFPKATNBL, showing PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17174591 that membrane localisation in the mRFP tagged protein is certainly dependent around the interaction with the PalMyrtagged protein. (JPG kb) Additional file Postembryonic tissue expression of C. elegans katanin genes mei, mei and FG Shown are fluorescence pictures of worms expressing a transcriptional GFP reporter below the c
ontrol from the indicated gene’s promoter, which stains the whole cell in which it can be expressed. DiI (red) costain ident.