R35807 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • N/A
    Additional name(s) for this target protein: ATP synthase subunit alpha
      Buy from Supplier

    86
    Millipore rabbit anti nek10
    a, 18S rRNA-normalized relative <t>NEK10</t> expression during ALI differentiation; n=1 ALI culture per timepoint. b-d , 18S rRNA-normalized relative expression of ciliated cell markers FOXJ1, DNAH5 ( b ), secretory cell marker SCGB1A1 ( c ), and basal cell marker KRT5 ( d ); n=1 ALI culture per timepoint. e-f, whole-mount immunofluorescence microscopy against SCGB1A1 ( e, upper panel), goblet cell marker MUC5AC ( e , lower panel), KRT5 ( f , upper panel), ciliated cell marker acetylated-α-tubulin ( f , lower panel); scale bars 100μm; bar graphs indicate fraction of surface epithelium occupied by marker-positive cells, n=4 per condition representative of 6 ALI differentiations, mean ±S.D. g, schematic depiction of bioinformatic NEK10 promoter (red) identification using indicated UCSC genome browser (hg19) tracks: CpG islands, H3K27-Ac, DNAse I hypersensitivity clusters, transcription factor (TF) chromatin immunoprecipitation sequencing (ChIP-seq). h, live GFP imaging of ALI cultures of the indicated genotypes and maturity, representative of 3 independent ALI differentiations; scale bars 200μm. i, gating strategy for FACS sorting of GFP-labeled ALI cultures, numbers indicate percentage gated cells per population.
    Rabbit Anti Nek10, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti nek10/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti nek10 - by Bioz Stars, 2024-02
    86/100 stars
      Buy from Supplier

    86
    Millipore anti acrbp rabbit
    a, 18S rRNA-normalized relative <t>NEK10</t> expression during ALI differentiation; n=1 ALI culture per timepoint. b-d , 18S rRNA-normalized relative expression of ciliated cell markers FOXJ1, DNAH5 ( b ), secretory cell marker SCGB1A1 ( c ), and basal cell marker KRT5 ( d ); n=1 ALI culture per timepoint. e-f, whole-mount immunofluorescence microscopy against SCGB1A1 ( e, upper panel), goblet cell marker MUC5AC ( e , lower panel), KRT5 ( f , upper panel), ciliated cell marker acetylated-α-tubulin ( f , lower panel); scale bars 100μm; bar graphs indicate fraction of surface epithelium occupied by marker-positive cells, n=4 per condition representative of 6 ALI differentiations, mean ±S.D. g, schematic depiction of bioinformatic NEK10 promoter (red) identification using indicated UCSC genome browser (hg19) tracks: CpG islands, H3K27-Ac, DNAse I hypersensitivity clusters, transcription factor (TF) chromatin immunoprecipitation sequencing (ChIP-seq). h, live GFP imaging of ALI cultures of the indicated genotypes and maturity, representative of 3 independent ALI differentiations; scale bars 200μm. i, gating strategy for FACS sorting of GFP-labeled ALI cultures, numbers indicate percentage gated cells per population.
    Anti Acrbp Rabbit, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti acrbp rabbit/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti acrbp rabbit - by Bioz Stars, 2024-02
    86/100 stars
      Buy from Supplier

    86
    Millipore rabbit polyclonal anti acrosin binding protein acrbp
    a, 18S rRNA-normalized relative <t>NEK10</t> expression during ALI differentiation; n=1 ALI culture per timepoint. b-d , 18S rRNA-normalized relative expression of ciliated cell markers FOXJ1, DNAH5 ( b ), secretory cell marker SCGB1A1 ( c ), and basal cell marker KRT5 ( d ); n=1 ALI culture per timepoint. e-f, whole-mount immunofluorescence microscopy against SCGB1A1 ( e, upper panel), goblet cell marker MUC5AC ( e , lower panel), KRT5 ( f , upper panel), ciliated cell marker acetylated-α-tubulin ( f , lower panel); scale bars 100μm; bar graphs indicate fraction of surface epithelium occupied by marker-positive cells, n=4 per condition representative of 6 ALI differentiations, mean ±S.D. g, schematic depiction of bioinformatic NEK10 promoter (red) identification using indicated UCSC genome browser (hg19) tracks: CpG islands, H3K27-Ac, DNAse I hypersensitivity clusters, transcription factor (TF) chromatin immunoprecipitation sequencing (ChIP-seq). h, live GFP imaging of ALI cultures of the indicated genotypes and maturity, representative of 3 independent ALI differentiations; scale bars 200μm. i, gating strategy for FACS sorting of GFP-labeled ALI cultures, numbers indicate percentage gated cells per population.
    Rabbit Polyclonal Anti Acrosin Binding Protein Acrbp, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti acrosin binding protein acrbp/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal anti acrosin binding protein acrbp - by Bioz Stars, 2024-02
    86/100 stars
      Buy from Supplier

    N/A
    Additional name(s) for this target protein: Ubiquitin-conjugating enzyme E2C
      Buy from Supplier

    Image Search Results


    a, 18S rRNA-normalized relative NEK10 expression during ALI differentiation; n=1 ALI culture per timepoint. b-d , 18S rRNA-normalized relative expression of ciliated cell markers FOXJ1, DNAH5 ( b ), secretory cell marker SCGB1A1 ( c ), and basal cell marker KRT5 ( d ); n=1 ALI culture per timepoint. e-f, whole-mount immunofluorescence microscopy against SCGB1A1 ( e, upper panel), goblet cell marker MUC5AC ( e , lower panel), KRT5 ( f , upper panel), ciliated cell marker acetylated-α-tubulin ( f , lower panel); scale bars 100μm; bar graphs indicate fraction of surface epithelium occupied by marker-positive cells, n=4 per condition representative of 6 ALI differentiations, mean ±S.D. g, schematic depiction of bioinformatic NEK10 promoter (red) identification using indicated UCSC genome browser (hg19) tracks: CpG islands, H3K27-Ac, DNAse I hypersensitivity clusters, transcription factor (TF) chromatin immunoprecipitation sequencing (ChIP-seq). h, live GFP imaging of ALI cultures of the indicated genotypes and maturity, representative of 3 independent ALI differentiations; scale bars 200μm. i, gating strategy for FACS sorting of GFP-labeled ALI cultures, numbers indicate percentage gated cells per population.

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, 18S rRNA-normalized relative NEK10 expression during ALI differentiation; n=1 ALI culture per timepoint. b-d , 18S rRNA-normalized relative expression of ciliated cell markers FOXJ1, DNAH5 ( b ), secretory cell marker SCGB1A1 ( c ), and basal cell marker KRT5 ( d ); n=1 ALI culture per timepoint. e-f, whole-mount immunofluorescence microscopy against SCGB1A1 ( e, upper panel), goblet cell marker MUC5AC ( e , lower panel), KRT5 ( f , upper panel), ciliated cell marker acetylated-α-tubulin ( f , lower panel); scale bars 100μm; bar graphs indicate fraction of surface epithelium occupied by marker-positive cells, n=4 per condition representative of 6 ALI differentiations, mean ±S.D. g, schematic depiction of bioinformatic NEK10 promoter (red) identification using indicated UCSC genome browser (hg19) tracks: CpG islands, H3K27-Ac, DNAse I hypersensitivity clusters, transcription factor (TF) chromatin immunoprecipitation sequencing (ChIP-seq). h, live GFP imaging of ALI cultures of the indicated genotypes and maturity, representative of 3 independent ALI differentiations; scale bars 200μm. i, gating strategy for FACS sorting of GFP-labeled ALI cultures, numbers indicate percentage gated cells per population.

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Expressing, Marker, Immunofluorescence, Microscopy, ChIP-sequencing, Imaging, Labeling

    a, Chest computed tomography (CT) imaging of proband 1 upon clinical presentation. Dashed line indicates level of cross-sectional imaging in right panel. Arrows highlight cystic bronchiectatic destruction of lung. b, transmission electron micrograph of proband 1 nasal biopsy specimen demonstrating normal radial ciliary ultrastructure, scale bars indicate 100nm. c, schematic depiction of 3’ terminus of NEK10 exon 15 and following intron, Sanger sequencing traces highlight G>C point mutation (red nucleotide) and high degree of conservation (red dashed box). d, 18S rRNA-normalized relative expression of indicated amplicons; n=3 independent lung tissue donors (controls), n=5 independently isolated lung regions (NEK10 G>C ), n=3 independently isolated HBEC lines for NEK10 G>C , n=1 for remaining samples, mean ±S.D. e, immunoblotting against the indicated proteins from cultured HBECs and ALI, NEK10 immunogen indicated, representative of 3 experiments. f, schematic representation of NEK10 cDNA sequencing results from indicated genotypes, common (yellow) and NEK10 G>C specific (red) residues indicated, canonical and cryptic splice donor motifs highlighted. g, immunoblotting after transient transfection of HEK293T cells with the indicated cDNAs, representative of 2 experiments. h, results of genome-wide linkage analysis incorporating individuals (n=15) highlighted with asterisks in ( , , ), peak bounded by marker SNPs rs13072262 and rs17798444, red line indicates LOD 3.3, equivalent to genome-wide p<0.05. Images in ( c ) and ( f ) generated from UCSC genome browser hg19 assembly ( http://genome.ucsc.edu ).

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, Chest computed tomography (CT) imaging of proband 1 upon clinical presentation. Dashed line indicates level of cross-sectional imaging in right panel. Arrows highlight cystic bronchiectatic destruction of lung. b, transmission electron micrograph of proband 1 nasal biopsy specimen demonstrating normal radial ciliary ultrastructure, scale bars indicate 100nm. c, schematic depiction of 3’ terminus of NEK10 exon 15 and following intron, Sanger sequencing traces highlight G>C point mutation (red nucleotide) and high degree of conservation (red dashed box). d, 18S rRNA-normalized relative expression of indicated amplicons; n=3 independent lung tissue donors (controls), n=5 independently isolated lung regions (NEK10 G>C ), n=3 independently isolated HBEC lines for NEK10 G>C , n=1 for remaining samples, mean ±S.D. e, immunoblotting against the indicated proteins from cultured HBECs and ALI, NEK10 immunogen indicated, representative of 3 experiments. f, schematic representation of NEK10 cDNA sequencing results from indicated genotypes, common (yellow) and NEK10 G>C specific (red) residues indicated, canonical and cryptic splice donor motifs highlighted. g, immunoblotting after transient transfection of HEK293T cells with the indicated cDNAs, representative of 2 experiments. h, results of genome-wide linkage analysis incorporating individuals (n=15) highlighted with asterisks in ( , , ), peak bounded by marker SNPs rs13072262 and rs17798444, red line indicates LOD 3.3, equivalent to genome-wide p<0.05. Images in ( c ) and ( f ) generated from UCSC genome browser hg19 assembly ( http://genome.ucsc.edu ).

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Computed Tomography, Imaging, Transmission Assay, Sequencing, Mutagenesis, Expressing, Isolation, Western Blot, Cell Culture, Transfection, Genome Wide, Marker, Generated

    a, 18S rRNA-normalized relative expression of indicated transcripts from FACS-sorted ALI cells; dashed line indicates expression level from unsorted mature ALI. b, confocal immunofluorescence of GFP in ciliated cells in NEK10p:eGFP ALI, representative of 2 independent ALI differentiations, scale bar 10μm. c, pseudocolored video microscopy of ALI of the indicated genotypes, representative of 3 independent ALI differentiations, scale bars 50μm. d, MCT (μOCT) of mature ALI of the indicated genotypes, n=485 (NEK10 WT ), 180 (NEK10 G>C ) pooled from 3 independent ALI differentiations, plot indicates median (center line), 25 th /75 th percentiles (box), 10 th /90 th (whiskers) percentiles, and remaining points (open circles). e, PCL (μOCT) of ALI of the indicated genotypes, n=11 (NEK10 WT ), 12 (NEK10 G>C ) pooled from 3 independent ALI differentiations, mean ±S.E.M. f, pseudocolored video microscopy of CRISPR/Cas9-edited ALI, representative fields from 3 independent ALI differentiations, scale bars 50μm. g, MCT of CRISPR/Cas9-edited ALI, n=361 (sgAAVS1), 131 (sgNEK10a), 59 (sgNEK10b), 104 (sgNEK10c) pooled from 3 independent ALI differentiations, plotted as in ( g ). h, PCL of CRISPR/Cas9-edited ALI, n=4 (sgAAVS1), 4 (sgNEK10a), 5 (sgNEK10b), 6 (sgNEK10c) pooled from 3 independent ALI differentiations, mean ±S.E.M. i, MCT of NEK10 G>C ALI expressing the indicated cDNAs, n=71 (no cDNA), 254 (NEK10 WT ), 129 (NEK10 K548R ), 1081 (NEK10 S684D ), pooled from 3 independent ALI differentiations, mean ±S.E.M. j, MCT of NEK10 WT ALI expressing the indicated cDNAs, n=1385 (FOXJ1:NEK10 K548R ), 1624 (FOXJ1:NEK10 WT ), 728 (FOXJ1:NEK10 S684D ), 401 (NEK10:NEK10 K548R ), 426 (NEK10:NEK10 WT ) pooled from 3 independent ALI differentiations, plotted as in ( g ). *p≤0.05, **p≤0.01, ****p≤0.0001

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, 18S rRNA-normalized relative expression of indicated transcripts from FACS-sorted ALI cells; dashed line indicates expression level from unsorted mature ALI. b, confocal immunofluorescence of GFP in ciliated cells in NEK10p:eGFP ALI, representative of 2 independent ALI differentiations, scale bar 10μm. c, pseudocolored video microscopy of ALI of the indicated genotypes, representative of 3 independent ALI differentiations, scale bars 50μm. d, MCT (μOCT) of mature ALI of the indicated genotypes, n=485 (NEK10 WT ), 180 (NEK10 G>C ) pooled from 3 independent ALI differentiations, plot indicates median (center line), 25 th /75 th percentiles (box), 10 th /90 th (whiskers) percentiles, and remaining points (open circles). e, PCL (μOCT) of ALI of the indicated genotypes, n=11 (NEK10 WT ), 12 (NEK10 G>C ) pooled from 3 independent ALI differentiations, mean ±S.E.M. f, pseudocolored video microscopy of CRISPR/Cas9-edited ALI, representative fields from 3 independent ALI differentiations, scale bars 50μm. g, MCT of CRISPR/Cas9-edited ALI, n=361 (sgAAVS1), 131 (sgNEK10a), 59 (sgNEK10b), 104 (sgNEK10c) pooled from 3 independent ALI differentiations, plotted as in ( g ). h, PCL of CRISPR/Cas9-edited ALI, n=4 (sgAAVS1), 4 (sgNEK10a), 5 (sgNEK10b), 6 (sgNEK10c) pooled from 3 independent ALI differentiations, mean ±S.E.M. i, MCT of NEK10 G>C ALI expressing the indicated cDNAs, n=71 (no cDNA), 254 (NEK10 WT ), 129 (NEK10 K548R ), 1081 (NEK10 S684D ), pooled from 3 independent ALI differentiations, mean ±S.E.M. j, MCT of NEK10 WT ALI expressing the indicated cDNAs, n=1385 (FOXJ1:NEK10 K548R ), 1624 (FOXJ1:NEK10 WT ), 728 (FOXJ1:NEK10 S684D ), 401 (NEK10:NEK10 K548R ), 426 (NEK10:NEK10 WT ) pooled from 3 independent ALI differentiations, plotted as in ( g ). *p≤0.05, **p≤0.01, ****p≤0.0001

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Expressing, Immunofluorescence, Microscopy, CRISPR

    a, quantitation of analysis in , mean ±S.D. b, kymographs of μOCT-based particle tracking from mature ALI, representative of 3 independent ALI differentiations. c, CBF (μOCT) of mature ALI of the indicated genotypes, n=27 (NEK10 WT ), 22 (NEK10 G>C ) pooled from 3 independent ALI differentiations, mean ±S.E.M. d, immunoblotting of mature ALI lysates after CRISPR/Cas9-mediated gene editing with the indicated sgRNAs, representative of 2 experiments; short (S) versus long (L) exposures indicated. e, quantitation of analysis in , mean ±S.D. f, CBF of mature ALI edited with the indicated sgRNAs, n=8 per condition pooled from 3 independent ALI differentiations, mean ±S.E.M. g, immunoblotting of mature ALI lysates transduced with the indicated cDNAs, representative of 2 experiments; short (S) versus long (L) exposures indicated. h, quantitation of analysis in ( i ), mean ±S.D. i, pseudocolored video microscopy of mature ALI transduced with the indicated cDNAs, representative fields from 3 independent ALI differentiations, scale bars 50μm. j, CBF of mature ALI transduced with the indicated cDNAs, n=4 per condition pooled from 3 independent ALI differentiations, mean ±S.E.M. *p≤0.05, **p≤0.01, ****p≤0.0001.

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, quantitation of analysis in , mean ±S.D. b, kymographs of μOCT-based particle tracking from mature ALI, representative of 3 independent ALI differentiations. c, CBF (μOCT) of mature ALI of the indicated genotypes, n=27 (NEK10 WT ), 22 (NEK10 G>C ) pooled from 3 independent ALI differentiations, mean ±S.E.M. d, immunoblotting of mature ALI lysates after CRISPR/Cas9-mediated gene editing with the indicated sgRNAs, representative of 2 experiments; short (S) versus long (L) exposures indicated. e, quantitation of analysis in , mean ±S.D. f, CBF of mature ALI edited with the indicated sgRNAs, n=8 per condition pooled from 3 independent ALI differentiations, mean ±S.E.M. g, immunoblotting of mature ALI lysates transduced with the indicated cDNAs, representative of 2 experiments; short (S) versus long (L) exposures indicated. h, quantitation of analysis in ( i ), mean ±S.D. i, pseudocolored video microscopy of mature ALI transduced with the indicated cDNAs, representative fields from 3 independent ALI differentiations, scale bars 50μm. j, CBF of mature ALI transduced with the indicated cDNAs, n=4 per condition pooled from 3 independent ALI differentiations, mean ±S.E.M. *p≤0.05, **p≤0.01, ****p≤0.0001.

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Quantitation Assay, Western Blot, CRISPR, Transduction, Microscopy

    a, schematic masking workflow for IFC morphological analysis. b, histogram of ciliary zone thickness of mature ALI MCCs of the indicated genotypes, n=4108 (NEK10 WT ), 3513 (NEK10 G>C ), shaded bars indicate medians ±0.25μm. c, histogram of ciliary area of mature ALI MCCs of the indicated genotypes, n=4108 (NEK10 WT ), 3513 (NEK10 G>C ). d, single cell images taken from the shaded regions in ( b ), scale bars 7μm. e, confocal maximum intensity projections (MIPs) of ALI of the indicated genotype and maturity following IF against Ac-α-tubulin, representative of 3 independent ALI differentiations, scale bars 25μm (left 4 panels) and 10μm (right 2 panels). f, confocal MIPs of mature ALI after IF against basal body marker centrin, dashed boxes mark full resolution regions in middle panels, scale bars 10μm (left 2 panels) and 1μm (middle 2 panels); column graph: centrin puncta per μm (mean ±S.D.) of ciliated cell surface area, n=71 cells and 10,855 puncta (NEK10 WT ), 38 cells and 5,369 puncta (NEK10 G>C ) pooled from 4 independent ALI differentiations. g, confocal MIPs of mature ALI after IF against PCP marker Vangl1, dashed boxes mark full resolution regions in right panel, scale bars 10μm (left panels) and 2.5μm (right panels), representative of 3 independent ALI differentiations. h, hematoxylin/eosin stained human large airway tissue; upper 3 samples taken from lung explants during transplantation for end-stage bronchiectasis due to the indicated etiologies, 4 th sample from patient undergoing resection for an unrelated diagnosis, scale bars 5μm. ****p≤0.0001.

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, schematic masking workflow for IFC morphological analysis. b, histogram of ciliary zone thickness of mature ALI MCCs of the indicated genotypes, n=4108 (NEK10 WT ), 3513 (NEK10 G>C ), shaded bars indicate medians ±0.25μm. c, histogram of ciliary area of mature ALI MCCs of the indicated genotypes, n=4108 (NEK10 WT ), 3513 (NEK10 G>C ). d, single cell images taken from the shaded regions in ( b ), scale bars 7μm. e, confocal maximum intensity projections (MIPs) of ALI of the indicated genotype and maturity following IF against Ac-α-tubulin, representative of 3 independent ALI differentiations, scale bars 25μm (left 4 panels) and 10μm (right 2 panels). f, confocal MIPs of mature ALI after IF against basal body marker centrin, dashed boxes mark full resolution regions in middle panels, scale bars 10μm (left 2 panels) and 1μm (middle 2 panels); column graph: centrin puncta per μm (mean ±S.D.) of ciliated cell surface area, n=71 cells and 10,855 puncta (NEK10 WT ), 38 cells and 5,369 puncta (NEK10 G>C ) pooled from 4 independent ALI differentiations. g, confocal MIPs of mature ALI after IF against PCP marker Vangl1, dashed boxes mark full resolution regions in right panel, scale bars 10μm (left panels) and 2.5μm (right panels), representative of 3 independent ALI differentiations. h, hematoxylin/eosin stained human large airway tissue; upper 3 samples taken from lung explants during transplantation for end-stage bronchiectasis due to the indicated etiologies, 4 th sample from patient undergoing resection for an unrelated diagnosis, scale bars 5μm. ****p≤0.0001.

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Marker, Staining, Transplantation Assay

    a, SEM of mature ALI of the indicated genotype, dashed boxes mark full resolution regions in right panel, scale bars 10μm (left panels) or 1μm (right panels), representative of 3 independent ALI differentiations. b, STEM of mature ALI of the indicated genotype after embedding and sectioning orthogonal to the epithelial surface, tick marks spaced at 1μm, representative of 3 independent ALI differentiations. c, representative negative stain EM grids prepared from purified cilia of the indicated genotypes, red scale bar indicates 1μm, representative of 2 independent ALI differentiations. d, histogram of ciliary length from purified cilia of the indicated genotypes, n=101 (NEK10 WT ), 102 (NEK10 G>C ) pooled from 2 independent ALI differentiations; inset: box-and-whisker plot of these data, center-line indicates median, box bounds 25 th and 75 th percentile, whiskers indicate 1.5*IQR, circles indicate outliers. e, cumulative distribution of phosphopeptides by log 2 fold change, previously identified motile ciliary proteins in red, all other detected proteins in black, sgNEK10b and sgNEK10c are independently targeting guide RNAs validated in . f, Table of ciliary genes by functional class with phosphopeptides depleted ≥2-fold upon NEK10 deletion. ****p≤0.0001

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, SEM of mature ALI of the indicated genotype, dashed boxes mark full resolution regions in right panel, scale bars 10μm (left panels) or 1μm (right panels), representative of 3 independent ALI differentiations. b, STEM of mature ALI of the indicated genotype after embedding and sectioning orthogonal to the epithelial surface, tick marks spaced at 1μm, representative of 3 independent ALI differentiations. c, representative negative stain EM grids prepared from purified cilia of the indicated genotypes, red scale bar indicates 1μm, representative of 2 independent ALI differentiations. d, histogram of ciliary length from purified cilia of the indicated genotypes, n=101 (NEK10 WT ), 102 (NEK10 G>C ) pooled from 2 independent ALI differentiations; inset: box-and-whisker plot of these data, center-line indicates median, box bounds 25 th and 75 th percentile, whiskers indicate 1.5*IQR, circles indicate outliers. e, cumulative distribution of phosphopeptides by log 2 fold change, previously identified motile ciliary proteins in red, all other detected proteins in black, sgNEK10b and sgNEK10c are independently targeting guide RNAs validated in . f, Table of ciliary genes by functional class with phosphopeptides depleted ≥2-fold upon NEK10 deletion. ****p≤0.0001

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Staining, Purification, Whisker Assay, Functional Assay

    a, SEM of mature ALI edited with the indicated sgRNAs, scale bars 100μm (upper panels) and 1μm (lower panels), representative of 2 independent ALI differentiations. b, immunoblotting against the indicated proteins from lysates generated from purified cilia (lanes 2, 4) or remaining de-ciliated mature ALI (lanes 1, 3), representative of 2 experiments. c , cumulative distribution of phosphopeptides by log 2 fold change between indicated conditions, solid (sgNEK10b) and dashed (sgNEK10c) red lines illustrate population of depleted phosphopeptides upon NEK10 deletion. d, table of gene ontology classes enriched among genes ( n =395) whose peptides are depleted >1.5 fold (log 2 ) after targeting with sgNEK10b, enrichment level, p -values, and false discovery rates (FDR) indicated. e, cumulative distribution of phosphopeptides by log 2 fold change, previously validated PCD in red and all other detected proteins in black, as in (4e). f , cumulative distribution of phosphopeptides by log 2 fold change, previously validated non-PCD ciliopathy loci in red and all other detected proteins in black, as in ( 4e ).

    Journal: Nature medicine

    Article Title: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance

    doi: 10.1038/s41591-019-0730-x

    Figure Lengend Snippet: a, SEM of mature ALI edited with the indicated sgRNAs, scale bars 100μm (upper panels) and 1μm (lower panels), representative of 2 independent ALI differentiations. b, immunoblotting against the indicated proteins from lysates generated from purified cilia (lanes 2, 4) or remaining de-ciliated mature ALI (lanes 1, 3), representative of 2 experiments. c , cumulative distribution of phosphopeptides by log 2 fold change between indicated conditions, solid (sgNEK10b) and dashed (sgNEK10c) red lines illustrate population of depleted phosphopeptides upon NEK10 deletion. d, table of gene ontology classes enriched among genes ( n =395) whose peptides are depleted >1.5 fold (log 2 ) after targeting with sgNEK10b, enrichment level, p -values, and false discovery rates (FDR) indicated. e, cumulative distribution of phosphopeptides by log 2 fold change, previously validated PCD in red and all other detected proteins in black, as in (4e). f , cumulative distribution of phosphopeptides by log 2 fold change, previously validated non-PCD ciliopathy loci in red and all other detected proteins in black, as in ( 4e ).

    Article Snippet: Primary antibodies and working dilutions were as follows: rabbit anti-NEK10 (Sigma HPA038941, lot R35857, 1:1000), mouse anti-NEK10 (Sigma WH0152110M1, lot 09058–1C9, 1:1000), rabbit anti-GAPDH (Abcam ab9485, 1:2500), mouse anti-FLAG M2 (Sigma F1804, lot SLBS3530V, 1:1000), rabbit anti-Raptor (Millipore 09–217, lot 3236353, 1:1000), mouse anti-β-actin (Santa Cruz sc-47778, lot K1718, 1:1000), mouse anti-acetylated α-tubulin (Sigma T7451, 1:1000).

    Techniques: Western Blot, Generated, Purification