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boc lys  (Chem Impex International)


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    Structured Review

    Chem Impex International boc lys
    Boc Lys, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/00647/Na-Boc-Ne-2-chloro-Z-L-lysine/pm29845860-115-0-20
    Average 96 stars, based on 2 article reviews
    boc lys - by Bioz Stars, 2026-09
    96/100 stars

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    Article Title: Using unnatural amino acids to probe the energetics of oxyanion hole hydrogen bonds in the ketosteroid isomerase active site.
    Article Snippet: Pyruvic acid (≥98%), p-dioxane (anhydrous, >99.8%) and Dowex 50WX8 cation exchange resin (100−200 mesh) were purchased from Acros Organics (Geel, Belgium); 3-chloro-L-tyrosine (97%) was purchased from Alfa Aesar (Ward Hill, MA, USA); di-tert-butyl dicarbonate (≥99%), trifluoroacetic acid (≥99.9%), O-(benzotriazol-1yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (≥99%), N2(tert-butoxycarbonyl)-N6-(((2-chlorobenzyl)oxy)carbonyl)-L-lysine (99%), O-benzyl-N-(tert-butoxycarbonyl)-L-threonine (99%) and N(tert-butoxycarbonyl)-L-leucine hydrate (99%) were purchased from Chem-Impex International (Wood Dale, IL, USA); N2-(tert-butoxycarbonyl)-N4-trityl-L-asparagine was purchased from AAPPTec (Louisville, KY, USA); S-trityl-β-mercaptopropionic acid was purchased from Peptides International (Louisville, KY, USA); and Boc-Leu-phenylacetamidomethyl resin and 1,4-dithio-DL-threitol (DTT) were purchased from Bachem (Torrance, CA, USA).

    Article Title: Metal Coordination to Ligand-Modified Peptide Nucleic Acid Triplexes.
    Article Snippet: A challenging goal in nanotechnology is the precise and programmable arrangement of specific elements in nanosystems in the three-dimensional space.. The use of ligand-modified nucleic acids represents an accurate and selective tool to achieve this goal when it comes to metal ion organization.. The synthesis of peptide nucleic acid (PNA) monomers that contain ligands instead of nucleobases makes possible the creation of metal-mediated alternative base pairs and triplets at specific locations in PNA duplexes and triplexes, respectively.

    Article Title: Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site
    Article Snippet: Pyruvic acid (≥98%), p-dioxane (anhydrous, >99.8%) and Dowex 50WX8 cation exchange resin (100−200 mesh) were purchased from Acros Organics (Geel, Belgium); 3-chloro-L-tyrosine (97%) was purchased from Alfa Aesar (Ward Hill, MA, USA); di-tert-butyl dicarbonate (≥99%), trifluoroacetic acid (≥99.9%), O-(benzotriazol-1yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (≥99%), N2(tert-butoxycarbonyl)-N6-(((2-chlorobenzyl)oxy)carbonyl)-L-lysine (99%), O-benzyl-N-(tert-butoxycarbonyl)-L-threonine (99%) and N(tert-butoxycarbonyl)-L-leucine hydrate (99%) were purchased from Chem-Impex International (Wood Dale, IL, USA); N2-(tert-butoxycarbonyl)-N4-trityl-L-asparagine was purchased from AAPPTec (Louisville, KY, USA); S-trityl-β-mercaptopropionic acid was purchased from Peptides International (Louisville, KY, USA); and Boc-Leu-phenylacetamidomethyl resin and 1,4-dithio-DL-threitol (DTT) were purchased from Bachem (Torrance, CA, USA).



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    Figure 1. Generation and characterization of <t>Dock4</t> (dedicator of cytokinesis 4)-deficient (D4−/−) mice. A, Schematic of DOCK4 genomic locus and targeting vector. Red triangles: LoxP sites. Blue ellipses: Frt (flippase recognition target) sites. Polymerase chain reaction with primer sets 5′F/5′R and 3′F/3′R was used to evaluate integration in embryonic stem cells. Polymerase chain reaction reactions showing the expected DNA fragments in one positive embryonic stem cell clone are shown. B, Genotyping of DOCK4-deficient mice using P1/P2 primer set. Primer locations are indicated. Expected DNA fragment sizes are shown for wt (wild type), heterozygous (wt/flx), and floxed animals (flx/flx). C, Schematic of Dock4 targeted allele after Cre recombination. Polymerase chain reaction using 5′Rec/3′Rec primer set shows the presence of the recombined allele in a D4−/− mouse. D, Western blot showing the absence of DOCK4 band in D4−/− mouse brain using N-terminal (left) and C-terminal (right) antibodies against DOCK4. E, Western blot analysis of D4−/− lungs. F and G, CD31 staining and vessel quantification of tumors from wt and D4−/− mice. F, B16F10 tumor implants were immunostained for CD31 (visualized with Alexa 488) and for nuclei (DAPI; left). CD31 masks (right) representing vascular structures were identified by image analysis as described in Methods. The scale bar shown as a white rectangle in the leftmost panel represents 100 µm and applies to all panels. G, The cross-sectional area of each CD31-expressing vessel in B16F10 tumors was quantified from CD31 masks, such as those illustrated in F, by image analysis as described in Methods. Frequency: the frequency of vessels in bins of 103 µm2 identified in each mouse relative to the total number of vessels identified in the same mouse. Cumulative: the fraction of vessels identified in each mouse with cross-sectional areas that were smaller than or equal to the bin values on the x axis. Individual data points are shown for each animal studied, and box plots represent the 25th, 50th, and 75th percentiles for the vessels in each bin from mice of each genotype. Con. indicates control; Cyto., cytoplasmic fraction; DT, diphtheria toxin; Insol., insoluble fraction; Memb., membrane fraction; P1 and P2, primers for polymerase chain reaction; PGK, phosphoglycerate kinase; and Pos., positive.
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    Image Search Results


    ( A ) Western blot analysis of RNF216 protein expression in GEVs and AEVs. ( B ) Western blot and quantitative analysis of RNF216 expression in neuronal cells ( n = 3 per group). ( C ) Lentiviral-mediated overexpression of RNF216 in neural stem cells. NSCs, neural stem cells. ( D ) RNF216 expression in Ad-Vec-NEVs and Ad-RNF216-NEVs assessed by Western blotting. ( E ) RNF216 levels in neuronal cells treated with Ad-Vec-NEVs or Ad-RNF216-NEVs, analyzed by Western blotting and quantification ( n = 3 per group). ( F ) Experimental grouping scheme. ( G ) BMS score evaluation of motor function in mice administered Ad-Vec-NEVs or Ad-RNF216-NEVs at 28 days postinjury ( n = 6 per group). ( H ) Rotarod performance comparison between the two groups with statistical analysis ( n = 6 per group). ( I and J ) Gait assessment via footprint analysis in both groups ( n = 6 per group). ( K ) Quantification of mechanical sensitivity using the von Frey filament test ( n = 6 per group). ( L ) NeuN immunofluorescence and statistical evaluation of neuronal survival ( n = 3 per group). ( M ) NF200 immunofluorescence and corresponding quantification of axonal integrity ( n = 3 per group). ( N and O ) Representative Western blots and quantitative analysis of ferroptosis-related proteins in the injured spinal cord (n = 3 per group). ( P to R ) RT-qPCR quantification of ferroptosis-associated gene expression in the injured spinal cord ( n = 3 per group). ( S to U ) Assessment of Fe 2+ concentration, GSH content, and MDA levels in spinal cord tissue ( n = 6 per group). ( V and W ) ROS detection and fluorescence intensity analysis in spinal cord sections via DHE staining ( n = 6 per group). ( X ) TUNEL staining and quantification of TUNEL-positive cells indicating cell death ( n = 6 per group). Note that n denotes the number of independent biological replicates.

    Journal: Science Advances

    Article Title: Athlete-derived extracellular vesicles protect against spinal cord injury via inhibition of neuronal ferroptosis

    doi: 10.1126/sciadv.adx7695

    Figure Lengend Snippet: ( A ) Western blot analysis of RNF216 protein expression in GEVs and AEVs. ( B ) Western blot and quantitative analysis of RNF216 expression in neuronal cells ( n = 3 per group). ( C ) Lentiviral-mediated overexpression of RNF216 in neural stem cells. NSCs, neural stem cells. ( D ) RNF216 expression in Ad-Vec-NEVs and Ad-RNF216-NEVs assessed by Western blotting. ( E ) RNF216 levels in neuronal cells treated with Ad-Vec-NEVs or Ad-RNF216-NEVs, analyzed by Western blotting and quantification ( n = 3 per group). ( F ) Experimental grouping scheme. ( G ) BMS score evaluation of motor function in mice administered Ad-Vec-NEVs or Ad-RNF216-NEVs at 28 days postinjury ( n = 6 per group). ( H ) Rotarod performance comparison between the two groups with statistical analysis ( n = 6 per group). ( I and J ) Gait assessment via footprint analysis in both groups ( n = 6 per group). ( K ) Quantification of mechanical sensitivity using the von Frey filament test ( n = 6 per group). ( L ) NeuN immunofluorescence and statistical evaluation of neuronal survival ( n = 3 per group). ( M ) NF200 immunofluorescence and corresponding quantification of axonal integrity ( n = 3 per group). ( N and O ) Representative Western blots and quantitative analysis of ferroptosis-related proteins in the injured spinal cord (n = 3 per group). ( P to R ) RT-qPCR quantification of ferroptosis-associated gene expression in the injured spinal cord ( n = 3 per group). ( S to U ) Assessment of Fe 2+ concentration, GSH content, and MDA levels in spinal cord tissue ( n = 6 per group). ( V and W ) ROS detection and fluorescence intensity analysis in spinal cord sections via DHE staining ( n = 6 per group). ( X ) TUNEL staining and quantification of TUNEL-positive cells indicating cell death ( n = 6 per group). Note that n denotes the number of independent biological replicates.

    Article Snippet: RNF216 − / − mice (C57BL/6NCya background) were generated by Cyagen Biosciences (Guangzhou, China) through CRISPR/Cas9-mediated deletion of exons 3 to 5 in the Rnf216 gene (NCBI RefSeq: NM_207110 ; Ensembl: ENSMUSG00000045078), which constitute ~37.3% of the coding sequence.

    Techniques: Western Blot, Expressing, Over Expression, Comparison, Immunofluorescence, Quantitative RT-PCR, Gene Expression, Concentration Assay, Fluorescence, Staining, TUNEL Assay

    ( A ) Representative peptide sequences. ( B ) Co-IP analysis confirmed endogenous RNF216-NOX1 interaction in neuronal cells. IgG, immunoglobulin G. ( C and D ) Exogenous expression of RNF216 and NOX1 in HEK 293T cells also demonstrated reciprocal binding under overexpression conditions. ( E ) Quantification of NOX1 mRNA levels in neuronal cells transfected with shCtrl or shRNF216 ( n = 3 per group). ( F and G ) Western blot analysis of RNF216 and NOX1 protein levels in shRNF216-transfected neuronal cells with or without MG132 treatment ( n = 3 per group). ( H ) Western blot detection of NOX1 and Flag-tagged RNF216 in HEK 293T cells transfected with increasing doses of WT or C688A RNF216 constructs. ( I and J ) Assessment of NOX1 protein stability following RNF216 overexpression in HEK 293T cells. ( K and L ) Evaluation of NOX1 protein stability after RNF216 knockdown in HEK 293T cells. Note that n denotes the number of independent biological replicates. CHX, cycloheximide. IB, Immunoblotting.

    Journal: Science Advances

    Article Title: Athlete-derived extracellular vesicles protect against spinal cord injury via inhibition of neuronal ferroptosis

    doi: 10.1126/sciadv.adx7695

    Figure Lengend Snippet: ( A ) Representative peptide sequences. ( B ) Co-IP analysis confirmed endogenous RNF216-NOX1 interaction in neuronal cells. IgG, immunoglobulin G. ( C and D ) Exogenous expression of RNF216 and NOX1 in HEK 293T cells also demonstrated reciprocal binding under overexpression conditions. ( E ) Quantification of NOX1 mRNA levels in neuronal cells transfected with shCtrl or shRNF216 ( n = 3 per group). ( F and G ) Western blot analysis of RNF216 and NOX1 protein levels in shRNF216-transfected neuronal cells with or without MG132 treatment ( n = 3 per group). ( H ) Western blot detection of NOX1 and Flag-tagged RNF216 in HEK 293T cells transfected with increasing doses of WT or C688A RNF216 constructs. ( I and J ) Assessment of NOX1 protein stability following RNF216 overexpression in HEK 293T cells. ( K and L ) Evaluation of NOX1 protein stability after RNF216 knockdown in HEK 293T cells. Note that n denotes the number of independent biological replicates. CHX, cycloheximide. IB, Immunoblotting.

    Article Snippet: RNF216 − / − mice (C57BL/6NCya background) were generated by Cyagen Biosciences (Guangzhou, China) through CRISPR/Cas9-mediated deletion of exons 3 to 5 in the Rnf216 gene (NCBI RefSeq: NM_207110 ; Ensembl: ENSMUSG00000045078), which constitute ~37.3% of the coding sequence.

    Techniques: Co-Immunoprecipitation Assay, Expressing, Binding Assay, Over Expression, Transfection, Western Blot, Construct, Knockdown

    ( A ) Neuronal cell lysates transfected with shCtrl or shRNF216 and treated with MG132 were subjected to immunoprecipitation with anti-NOX1, followed by immunoblotting using the indicated antibodies. ( B ) Quantification of Ub-conjugated NOX1 (Ub-NOX1) levels ( n = 3 per group). ( C ) Analysis of NOX1 ubiquitination in neuronal cells overexpressing either WT RNF216 or its C688A mutant. ( D ) Quantification of Ub-NOX1 in response to RNF216 or C688A overexpression ( n = 3 per group). ( E ) Immunoprecipitation of spinal cord lysates postinjury with anti-NOX1, followed by immunoblotting for ubiquitin and NOX1 in the indicated mouse groups. ( F ) Quantification of relative Ub-NOX1 levels from (E) ( n = 3 per group). ( G ) Comparative analysis of NOX1 ubiquitination in spinal cord lysates from both mouse groups postinjury via immunoprecipitation and Western blotting. ( H ) Quantification of Ub-NOX1 from (G) ( n = 3 per group). ( I ) HEK 293T cells were cotransfected with Flag-RNF216, Myc-NOX1, and HA-tagged ubiquitin variants (WT, K48 only, and K63 only) to evaluate linkage specificity in NOX1 ubiquitination. ( J ) HEK 293T cells transfected with Ub WT or Ub K63R were cultured with either shCtrl or shRNF216. Lysates were analyzed by Western blotting with anti-NOX1 and anti-RNF216. Note that n denotes the number of independent biological replicates.

    Journal: Science Advances

    Article Title: Athlete-derived extracellular vesicles protect against spinal cord injury via inhibition of neuronal ferroptosis

    doi: 10.1126/sciadv.adx7695

    Figure Lengend Snippet: ( A ) Neuronal cell lysates transfected with shCtrl or shRNF216 and treated with MG132 were subjected to immunoprecipitation with anti-NOX1, followed by immunoblotting using the indicated antibodies. ( B ) Quantification of Ub-conjugated NOX1 (Ub-NOX1) levels ( n = 3 per group). ( C ) Analysis of NOX1 ubiquitination in neuronal cells overexpressing either WT RNF216 or its C688A mutant. ( D ) Quantification of Ub-NOX1 in response to RNF216 or C688A overexpression ( n = 3 per group). ( E ) Immunoprecipitation of spinal cord lysates postinjury with anti-NOX1, followed by immunoblotting for ubiquitin and NOX1 in the indicated mouse groups. ( F ) Quantification of relative Ub-NOX1 levels from (E) ( n = 3 per group). ( G ) Comparative analysis of NOX1 ubiquitination in spinal cord lysates from both mouse groups postinjury via immunoprecipitation and Western blotting. ( H ) Quantification of Ub-NOX1 from (G) ( n = 3 per group). ( I ) HEK 293T cells were cotransfected with Flag-RNF216, Myc-NOX1, and HA-tagged ubiquitin variants (WT, K48 only, and K63 only) to evaluate linkage specificity in NOX1 ubiquitination. ( J ) HEK 293T cells transfected with Ub WT or Ub K63R were cultured with either shCtrl or shRNF216. Lysates were analyzed by Western blotting with anti-NOX1 and anti-RNF216. Note that n denotes the number of independent biological replicates.

    Article Snippet: RNF216 − / − mice (C57BL/6NCya background) were generated by Cyagen Biosciences (Guangzhou, China) through CRISPR/Cas9-mediated deletion of exons 3 to 5 in the Rnf216 gene (NCBI RefSeq: NM_207110 ; Ensembl: ENSMUSG00000045078), which constitute ~37.3% of the coding sequence.

    Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Mutagenesis, Over Expression, Cell Culture

    ( A ) Experimental subgrouping information. ( B ) BMS scores assessed 28 days postinjury in mice treated with Ad-RNF216-NEVs + Ad-Vec or Ad-RNF216-NEVs + Ad-NOX1 ( n = 6 per group). ( C ) Quantitative analysis of motor coordination using the rotarod test ( n = 6 per group). ( D and E ) Gait evaluation via footprint analysis in both treatment groups ( n = 6 per group). ( F ) Mechanical sensitivity assessment using the von Frey filament test ( n = 6 per group). ( G ) NeuN immunofluorescence staining and corresponding quantification of neuronal survival ( n = 3 per group). ( H ) NF200 immunofluorescence staining and quantification of axonal integrity ( n = 3 per group). ( I and J ) Representative Western blot images and statistical quantification of ferroptosis-related proteins in the injured spinal cord ( n = 3 per group). ( K ) RT-qPCR quantification of mRNA expression levels of ferroptosis markers in spinal cord tissue ( n = 3 per group). ( L to N ) Quantification of Fe 2+ concentration, GSH content, and MDA levels in spinal cord homogenates ( n = 6 per group). ( O and P ) ROS detection and fluorescence quantification using DHE staining in spinal cord sections ( n = 6 per group). ( Q and R ) Cell damage assessment and quantification of TUNEL-positive cells using TUNEL staining ( n = 6 per group). Note that n denotes the number of independent biological replicates.

    Journal: Science Advances

    Article Title: Athlete-derived extracellular vesicles protect against spinal cord injury via inhibition of neuronal ferroptosis

    doi: 10.1126/sciadv.adx7695

    Figure Lengend Snippet: ( A ) Experimental subgrouping information. ( B ) BMS scores assessed 28 days postinjury in mice treated with Ad-RNF216-NEVs + Ad-Vec or Ad-RNF216-NEVs + Ad-NOX1 ( n = 6 per group). ( C ) Quantitative analysis of motor coordination using the rotarod test ( n = 6 per group). ( D and E ) Gait evaluation via footprint analysis in both treatment groups ( n = 6 per group). ( F ) Mechanical sensitivity assessment using the von Frey filament test ( n = 6 per group). ( G ) NeuN immunofluorescence staining and corresponding quantification of neuronal survival ( n = 3 per group). ( H ) NF200 immunofluorescence staining and quantification of axonal integrity ( n = 3 per group). ( I and J ) Representative Western blot images and statistical quantification of ferroptosis-related proteins in the injured spinal cord ( n = 3 per group). ( K ) RT-qPCR quantification of mRNA expression levels of ferroptosis markers in spinal cord tissue ( n = 3 per group). ( L to N ) Quantification of Fe 2+ concentration, GSH content, and MDA levels in spinal cord homogenates ( n = 6 per group). ( O and P ) ROS detection and fluorescence quantification using DHE staining in spinal cord sections ( n = 6 per group). ( Q and R ) Cell damage assessment and quantification of TUNEL-positive cells using TUNEL staining ( n = 6 per group). Note that n denotes the number of independent biological replicates.

    Article Snippet: RNF216 − / − mice (C57BL/6NCya background) were generated by Cyagen Biosciences (Guangzhou, China) through CRISPR/Cas9-mediated deletion of exons 3 to 5 in the Rnf216 gene (NCBI RefSeq: NM_207110 ; Ensembl: ENSMUSG00000045078), which constitute ~37.3% of the coding sequence.

    Techniques: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Expressing, Concentration Assay, Fluorescence, TUNEL Assay

    AEVs reduce mitochondrial damage and ferroptosis in neuronal cells by modulating ferroptosis-related pathways. RNF216 in AEVs enhances the ubiquitination and degradation of NOX1, reducing oxidative stress and ferroptotic cell death, thereby supporting functional recovery after SCI.

    Journal: Science Advances

    Article Title: Athlete-derived extracellular vesicles protect against spinal cord injury via inhibition of neuronal ferroptosis

    doi: 10.1126/sciadv.adx7695

    Figure Lengend Snippet: AEVs reduce mitochondrial damage and ferroptosis in neuronal cells by modulating ferroptosis-related pathways. RNF216 in AEVs enhances the ubiquitination and degradation of NOX1, reducing oxidative stress and ferroptotic cell death, thereby supporting functional recovery after SCI.

    Article Snippet: RNF216 − / − mice (C57BL/6NCya background) were generated by Cyagen Biosciences (Guangzhou, China) through CRISPR/Cas9-mediated deletion of exons 3 to 5 in the Rnf216 gene (NCBI RefSeq: NM_207110 ; Ensembl: ENSMUSG00000045078), which constitute ~37.3% of the coding sequence.

    Techniques: Ubiquitin Proteomics, Functional Assay

    Figure 1. Generation and characterization of Dock4 (dedicator of cytokinesis 4)-deficient (D4−/−) mice. A, Schematic of DOCK4 genomic locus and targeting vector. Red triangles: LoxP sites. Blue ellipses: Frt (flippase recognition target) sites. Polymerase chain reaction with primer sets 5′F/5′R and 3′F/3′R was used to evaluate integration in embryonic stem cells. Polymerase chain reaction reactions showing the expected DNA fragments in one positive embryonic stem cell clone are shown. B, Genotyping of DOCK4-deficient mice using P1/P2 primer set. Primer locations are indicated. Expected DNA fragment sizes are shown for wt (wild type), heterozygous (wt/flx), and floxed animals (flx/flx). C, Schematic of Dock4 targeted allele after Cre recombination. Polymerase chain reaction using 5′Rec/3′Rec primer set shows the presence of the recombined allele in a D4−/− mouse. D, Western blot showing the absence of DOCK4 band in D4−/− mouse brain using N-terminal (left) and C-terminal (right) antibodies against DOCK4. E, Western blot analysis of D4−/− lungs. F and G, CD31 staining and vessel quantification of tumors from wt and D4−/− mice. F, B16F10 tumor implants were immunostained for CD31 (visualized with Alexa 488) and for nuclei (DAPI; left). CD31 masks (right) representing vascular structures were identified by image analysis as described in Methods. The scale bar shown as a white rectangle in the leftmost panel represents 100 µm and applies to all panels. G, The cross-sectional area of each CD31-expressing vessel in B16F10 tumors was quantified from CD31 masks, such as those illustrated in F, by image analysis as described in Methods. Frequency: the frequency of vessels in bins of 103 µm2 identified in each mouse relative to the total number of vessels identified in the same mouse. Cumulative: the fraction of vessels identified in each mouse with cross-sectional areas that were smaller than or equal to the bin values on the x axis. Individual data points are shown for each animal studied, and box plots represent the 25th, 50th, and 75th percentiles for the vessels in each bin from mice of each genotype. Con. indicates control; Cyto., cytoplasmic fraction; DT, diphtheria toxin; Insol., insoluble fraction; Memb., membrane fraction; P1 and P2, primers for polymerase chain reaction; PGK, phosphoglycerate kinase; and Pos., positive.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function

    doi: 10.1161/atvbaha.122.317565

    Figure Lengend Snippet: Figure 1. Generation and characterization of Dock4 (dedicator of cytokinesis 4)-deficient (D4−/−) mice. A, Schematic of DOCK4 genomic locus and targeting vector. Red triangles: LoxP sites. Blue ellipses: Frt (flippase recognition target) sites. Polymerase chain reaction with primer sets 5′F/5′R and 3′F/3′R was used to evaluate integration in embryonic stem cells. Polymerase chain reaction reactions showing the expected DNA fragments in one positive embryonic stem cell clone are shown. B, Genotyping of DOCK4-deficient mice using P1/P2 primer set. Primer locations are indicated. Expected DNA fragment sizes are shown for wt (wild type), heterozygous (wt/flx), and floxed animals (flx/flx). C, Schematic of Dock4 targeted allele after Cre recombination. Polymerase chain reaction using 5′Rec/3′Rec primer set shows the presence of the recombined allele in a D4−/− mouse. D, Western blot showing the absence of DOCK4 band in D4−/− mouse brain using N-terminal (left) and C-terminal (right) antibodies against DOCK4. E, Western blot analysis of D4−/− lungs. F and G, CD31 staining and vessel quantification of tumors from wt and D4−/− mice. F, B16F10 tumor implants were immunostained for CD31 (visualized with Alexa 488) and for nuclei (DAPI; left). CD31 masks (right) representing vascular structures were identified by image analysis as described in Methods. The scale bar shown as a white rectangle in the leftmost panel represents 100 µm and applies to all panels. G, The cross-sectional area of each CD31-expressing vessel in B16F10 tumors was quantified from CD31 masks, such as those illustrated in F, by image analysis as described in Methods. Frequency: the frequency of vessels in bins of 103 µm2 identified in each mouse relative to the total number of vessels identified in the same mouse. Cumulative: the fraction of vessels identified in each mouse with cross-sectional areas that were smaller than or equal to the bin values on the x axis. Individual data points are shown for each animal studied, and box plots represent the 25th, 50th, and 75th percentiles for the vessels in each bin from mice of each genotype. Con. indicates control; Cyto., cytoplasmic fraction; DT, diphtheria toxin; Insol., insoluble fraction; Memb., membrane fraction; P1 and P2, primers for polymerase chain reaction; PGK, phosphoglycerate kinase; and Pos., positive.

    Article Snippet: DOI: 10.1161/ATVBAHA.122.317565 and probed with primary antibodies against DOCK4 (1:500; catalog number IHC-00647; Bethyl), RhoA (Ras homolog family member A; 1:500; sc-418; Santa Cruz), actin (1:4000; A3853; Sigma), tubulin (1:5000; T5168; Sigma), phospho-MLC (myosin light chain; Ser19; 1:1000; 3671; Cell Signaling Technologies), and total MLC (1:1000; 8505; Cell Signaling Technologies).

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Western Blot, Staining, Expressing, Control, Membrane

    Figure 2. Enhanced vascular permeability in DOCK4 (dedicator of cytokinesis 4)-deficient (D4−/−) mice. A, A representative image of buffer-perfused lungs harvested from wt (wild type) and D4−/− mice. B, Assessment of lung hemorrhage score (left). Plot shows fraction of bronchioles with peribronchial hemorrhage (top) and diffuse hemorrhage (bottom) score. (Continued )

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function

    doi: 10.1161/atvbaha.122.317565

    Figure Lengend Snippet: Figure 2. Enhanced vascular permeability in DOCK4 (dedicator of cytokinesis 4)-deficient (D4−/−) mice. A, A representative image of buffer-perfused lungs harvested from wt (wild type) and D4−/− mice. B, Assessment of lung hemorrhage score (left). Plot shows fraction of bronchioles with peribronchial hemorrhage (top) and diffuse hemorrhage (bottom) score. (Continued )

    Article Snippet: DOI: 10.1161/ATVBAHA.122.317565 and probed with primary antibodies against DOCK4 (1:500; catalog number IHC-00647; Bethyl), RhoA (Ras homolog family member A; 1:500; sc-418; Santa Cruz), actin (1:4000; A3853; Sigma), tubulin (1:5000; T5168; Sigma), phospho-MLC (myosin light chain; Ser19; 1:1000; 3671; Cell Signaling Technologies), and total MLC (1:1000; 8505; Cell Signaling Technologies).

    Techniques: Permeability

    Figure 3. DOCK4 (dedicator of cytokinesis 4) silencing in human lung endothelial cells increases basal permeability. A, Human pulmonary artery endothelial cells (HPAECs) were electroporated with control (Ctrl) or DOCK4 siRNA and seeded on gold- plated electrodes for 48 h. Cells were serum starved for 2 h after which endothelial barrier function was determined by measuring transendothelial electrical resistance over time (in hours) before and after addition of 1 µmol/L S1P (sphingosine-1-phosphate). Data are mean±SEM; 2-way ANOVA multiple comparison (Bonferroni). B, Western blot of DOCK4 in siRNA cells. C, mRNA levels of S1PR1 (S1P receptor; left) and DOCK4 (right) in DOCK4 knockdown HPAEC using 2 independent DOCK4 shRNAs. Data are mean±SEM fold increase vs levels in HPAECs transduced with shRNA control lentivirus (Ctrl); Welch t test. D, mRNA levels of indicated guanine exchange factors in HPAECs. Data are mean±SEM; 1-way ANOVA, Tukey multiple comparisons. E, HPAECs electroporated with Ctrl or DOCK4 siRNA were plated on 6-well plate. After 48 h, cells were starved for 5 h, stimulated with 1 µM S1P for different times, and lysates were used to assess Rac-1 activity. Data are mean±SEM; 2-way ANOVA, Tukey multiple comparisons. F, HPAECs transfected with Ctrl and DOCK4 siRNA were plated on glass coverslips for 48 h. Cells were serum starved for 4 h followed by treatment with 1 µmol/L S1P for the indicated times in minutes. Cells were fixed and stained for DOCK4 (green; n=3). Bar, 20 µm. G, HPAECs were treated with S1P or vehicle control for 1 min, and DOCK4 levels in equal amounts of protein from each of the obtained fractions (cytosolic, membrane, and insoluble cytoskeletal/nuclear) were analyzed by Western blot (left). Western blot quantification of DOCK4 levels after S1P treatment (right). Data are mean fold increase in S1P treated vs control cells ±SD; Mann-Whitney U test. Tiam indicates T-cell lymphoma invasion and metastasis.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function

    doi: 10.1161/atvbaha.122.317565

    Figure Lengend Snippet: Figure 3. DOCK4 (dedicator of cytokinesis 4) silencing in human lung endothelial cells increases basal permeability. A, Human pulmonary artery endothelial cells (HPAECs) were electroporated with control (Ctrl) or DOCK4 siRNA and seeded on gold- plated electrodes for 48 h. Cells were serum starved for 2 h after which endothelial barrier function was determined by measuring transendothelial electrical resistance over time (in hours) before and after addition of 1 µmol/L S1P (sphingosine-1-phosphate). Data are mean±SEM; 2-way ANOVA multiple comparison (Bonferroni). B, Western blot of DOCK4 in siRNA cells. C, mRNA levels of S1PR1 (S1P receptor; left) and DOCK4 (right) in DOCK4 knockdown HPAEC using 2 independent DOCK4 shRNAs. Data are mean±SEM fold increase vs levels in HPAECs transduced with shRNA control lentivirus (Ctrl); Welch t test. D, mRNA levels of indicated guanine exchange factors in HPAECs. Data are mean±SEM; 1-way ANOVA, Tukey multiple comparisons. E, HPAECs electroporated with Ctrl or DOCK4 siRNA were plated on 6-well plate. After 48 h, cells were starved for 5 h, stimulated with 1 µM S1P for different times, and lysates were used to assess Rac-1 activity. Data are mean±SEM; 2-way ANOVA, Tukey multiple comparisons. F, HPAECs transfected with Ctrl and DOCK4 siRNA were plated on glass coverslips for 48 h. Cells were serum starved for 4 h followed by treatment with 1 µmol/L S1P for the indicated times in minutes. Cells were fixed and stained for DOCK4 (green; n=3). Bar, 20 µm. G, HPAECs were treated with S1P or vehicle control for 1 min, and DOCK4 levels in equal amounts of protein from each of the obtained fractions (cytosolic, membrane, and insoluble cytoskeletal/nuclear) were analyzed by Western blot (left). Western blot quantification of DOCK4 levels after S1P treatment (right). Data are mean fold increase in S1P treated vs control cells ±SD; Mann-Whitney U test. Tiam indicates T-cell lymphoma invasion and metastasis.

    Article Snippet: DOI: 10.1161/ATVBAHA.122.317565 and probed with primary antibodies against DOCK4 (1:500; catalog number IHC-00647; Bethyl), RhoA (Ras homolog family member A; 1:500; sc-418; Santa Cruz), actin (1:4000; A3853; Sigma), tubulin (1:5000; T5168; Sigma), phospho-MLC (myosin light chain; Ser19; 1:1000; 3671; Cell Signaling Technologies), and total MLC (1:1000; 8505; Cell Signaling Technologies).

    Techniques: Permeability, Control, Comparison, Western Blot, Knockdown, Transduction, shRNA, Activity Assay, Transfection, Staining, Membrane, MANN-WHITNEY

    Figure 4. DOCK4 (dedicator of cytokinesis 4) maintains the balance between Rho (Ras homology)/Rac-1 GTPases. A, DOCK4 depletion induces actin stress fibers. Human pulmonary artery endothelial cells (HPAECs) were transduced with control (Cntrl) shRNA or 2 DOCK4 D4(1) and D4(2) shRNA clones for 72 h. Cells were fixed and stained with phalloidin 568, and mean phalloidin intensity was measured. Data are mean±SEM; 1-way ANOVA, Tukey multiple comparisons. B, Representative Western blot of DOCK4 and RhoA (Ras homolog family member A) in shRNA cells. C, HPAECs transduced with Cntrl and D4 shRNAs for 72 h were starved for 4 to 5 h, and lysates were used to assess RhoA GTPase activity using an ELISA assay. Data are mean±SEM; 1-way ANOVA, Tukey multiple comparisons (n=4). D, Cntrl and DOCK4 shRNA HPAECs treated with vehicle (veh) or ROCK (Rho-associated protein kinase) inhibitor (Y27632) 10 µM for 10 min were fixed, stained with Phalloidin Alexa 568, and quantified for mean phalloidin intensity. Data are mean±SEM; 2-way ANOVA, Tukey multiple comparisons. E, DOCK4 deficiency results in increased MLC (myosin light chain) phosphorylation. HPAECs were transduced with control or DOCK4 shRNA lentiviruses as in A. After 72 h, the levels of phosphorylated MLC were assessed by Western blot analysis. Total MLC and actin served as loading controls. Data are mean±SD; 1-way ANOVA and Tukey multiple comparisons. F, HPAECs were transduced with control or DOCK4 ShRNA lentiviruses as in A. After 72 h, cells were starved for 4 h and Rac activity was measured after treatment with 1 µM S1P (sphingosine-1-phosphate) at the indicated times using an ELISA assay. Data are mean±SD; 2-way ANOVA and Tukey multiple comparisons.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function

    doi: 10.1161/atvbaha.122.317565

    Figure Lengend Snippet: Figure 4. DOCK4 (dedicator of cytokinesis 4) maintains the balance between Rho (Ras homology)/Rac-1 GTPases. A, DOCK4 depletion induces actin stress fibers. Human pulmonary artery endothelial cells (HPAECs) were transduced with control (Cntrl) shRNA or 2 DOCK4 D4(1) and D4(2) shRNA clones for 72 h. Cells were fixed and stained with phalloidin 568, and mean phalloidin intensity was measured. Data are mean±SEM; 1-way ANOVA, Tukey multiple comparisons. B, Representative Western blot of DOCK4 and RhoA (Ras homolog family member A) in shRNA cells. C, HPAECs transduced with Cntrl and D4 shRNAs for 72 h were starved for 4 to 5 h, and lysates were used to assess RhoA GTPase activity using an ELISA assay. Data are mean±SEM; 1-way ANOVA, Tukey multiple comparisons (n=4). D, Cntrl and DOCK4 shRNA HPAECs treated with vehicle (veh) or ROCK (Rho-associated protein kinase) inhibitor (Y27632) 10 µM for 10 min were fixed, stained with Phalloidin Alexa 568, and quantified for mean phalloidin intensity. Data are mean±SEM; 2-way ANOVA, Tukey multiple comparisons. E, DOCK4 deficiency results in increased MLC (myosin light chain) phosphorylation. HPAECs were transduced with control or DOCK4 shRNA lentiviruses as in A. After 72 h, the levels of phosphorylated MLC were assessed by Western blot analysis. Total MLC and actin served as loading controls. Data are mean±SD; 1-way ANOVA and Tukey multiple comparisons. F, HPAECs were transduced with control or DOCK4 ShRNA lentiviruses as in A. After 72 h, cells were starved for 4 h and Rac activity was measured after treatment with 1 µM S1P (sphingosine-1-phosphate) at the indicated times using an ELISA assay. Data are mean±SD; 2-way ANOVA and Tukey multiple comparisons.

    Article Snippet: DOI: 10.1161/ATVBAHA.122.317565 and probed with primary antibodies against DOCK4 (1:500; catalog number IHC-00647; Bethyl), RhoA (Ras homolog family member A; 1:500; sc-418; Santa Cruz), actin (1:4000; A3853; Sigma), tubulin (1:5000; T5168; Sigma), phospho-MLC (myosin light chain; Ser19; 1:1000; 3671; Cell Signaling Technologies), and total MLC (1:1000; 8505; Cell Signaling Technologies).

    Techniques: Transduction, Control, shRNA, Clone Assay, Staining, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

    Figure 5. DOCK4 (dedicator of cytokinesis 4) expression in DOCK4 depleted endothelial cells restores barrier function. A, Transduction of control (Cntrl) or D4 shRNA endothelial monolayers with wild-type DOCK4 or LacZ (beta-galactosidase) adenovirus as indicated. Monolayers transduced with DOCK4 resulted in DOCK4 protein expression. Representative Western blot is shown. B, VE-cadherin staining (arrow) of Cntrl and D4 shRNA cells transduced with LacZ or DOCK4 adenovirus is indicated. C, Human pulmonary artery endothelial cells were transduced with Cntrl or DOCK4 shRNA clones for 48 h. Cells were trypsinized and plated on electrodes to allow monolayers to form. Wells were then transduced with LacZ or DOCK4 adenovirus for 24 h. Basal resistance was measured after 72 h. Data are mean±SEM; 2-way ANOVA with Tukey multiple comparisons. D, Cntrl or DOCK4 shRNA cells as in C were transduced with LacZ or DOCK4 adenovirus, respectively. After 24 h, cells were starved for 2 h and transendothelial electrical resistance was examined before and after stimulation with 1 µmol/L S1P (sphingosine-1- phosphate). Each experimental sample was normalized (N) to its own baseline (−S1P). Data are mean±SEM; 2-way ANOVA with Tukey multiple comparisons.

    Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

    Article Title: DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function

    doi: 10.1161/atvbaha.122.317565

    Figure Lengend Snippet: Figure 5. DOCK4 (dedicator of cytokinesis 4) expression in DOCK4 depleted endothelial cells restores barrier function. A, Transduction of control (Cntrl) or D4 shRNA endothelial monolayers with wild-type DOCK4 or LacZ (beta-galactosidase) adenovirus as indicated. Monolayers transduced with DOCK4 resulted in DOCK4 protein expression. Representative Western blot is shown. B, VE-cadherin staining (arrow) of Cntrl and D4 shRNA cells transduced with LacZ or DOCK4 adenovirus is indicated. C, Human pulmonary artery endothelial cells were transduced with Cntrl or DOCK4 shRNA clones for 48 h. Cells were trypsinized and plated on electrodes to allow monolayers to form. Wells were then transduced with LacZ or DOCK4 adenovirus for 24 h. Basal resistance was measured after 72 h. Data are mean±SEM; 2-way ANOVA with Tukey multiple comparisons. D, Cntrl or DOCK4 shRNA cells as in C were transduced with LacZ or DOCK4 adenovirus, respectively. After 24 h, cells were starved for 2 h and transendothelial electrical resistance was examined before and after stimulation with 1 µmol/L S1P (sphingosine-1- phosphate). Each experimental sample was normalized (N) to its own baseline (−S1P). Data are mean±SEM; 2-way ANOVA with Tukey multiple comparisons.

    Article Snippet: DOI: 10.1161/ATVBAHA.122.317565 and probed with primary antibodies against DOCK4 (1:500; catalog number IHC-00647; Bethyl), RhoA (Ras homolog family member A; 1:500; sc-418; Santa Cruz), actin (1:4000; A3853; Sigma), tubulin (1:5000; T5168; Sigma), phospho-MLC (myosin light chain; Ser19; 1:1000; 3671; Cell Signaling Technologies), and total MLC (1:1000; 8505; Cell Signaling Technologies).

    Techniques: Expressing, Transduction, Control, shRNA, Western Blot, Staining, Clone Assay