primary antibodies anti talin1  (Bio-Rad)

Journal: JCI Insight

Article Title: Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

doi: 10.1172/jci.insight.173664

Figure Lengend Snippet: ( A ) Representative confocal 3D images of VE-cadherin (green) immunostained confluent monolayers of heterozygous-talin1 primary ECs transfected with full-length talin1 protein (EC-Tln WT ) or SCLS- TLN1 mutant lacking the 21 aa of exon 54 (EC-Tln Δex54 ) or the talin1 ABS3 mutation, R2510A (EC-Tln ABS3 ). Nuclei were stained with DAPI (blue). ( B ) Color scaling of VE-cadherin, whereby the red color is the highest continuous staining area (>70 μm 2 ), decreasing to smaller areas marked by different colors until it reaches the lowest measurements, which are of blue-violet color (<30 μm 2 ), analyzed by IMARIS. Scale bars: 10 μm. ( C ) Graph displays the quantification of the continuous junctional area, as represented by the percentage of staining surfaces greater than 30 μm 2 versus the total staining area. Data represent the mean area per field of monolayer. n fields of view analyzed: EC-Tln WT = 27; EC-Tln Δex54 = 21; EC-Tln ABS3 = 22. Red dots represent the mean ± SEM of 3 independent experiment. **** P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. ( D ) Graph displays the distribution of 3 different indexes of junctional fragments as a percentage of the total staining surfaces. Data represent the mean ± SEM of 3 independent experiments. n of field of views analyzed: EC-Tln WT = 25; EC-Tln Δex54 = 22; EC-Tln ABS3 = 22.

Article Snippet: Primary antibodies anti-talin1 (clone 97H6, Bio-Rad, MCA4770, 1:500), anti-talin2 (custom made, 1:50; ref. ), anti-paxillin (BD Biosciences, 610052, 1:200), anti–p-Tyr118-paxillin (Cell Signaling Technology, 2541, 1:200), anti-vinculin (Sigma-Aldrich, V9264, 1:400), anti–p-Tyr397-FAK (Biosource, 44624G, 1:100), or anti–integrin-β1 (clone 9EG7, BD Biosciences, 550531) were added on the coverslips in 0.1% BSA/PBS blocking buffer and incubated overnight at 4°C.

Techniques: Transfection, Mutagenesis, Staining, Comparison

Journal: JCI Insight

Article Title: Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

doi: 10.1172/jci.insight.173664

Figure Lengend Snippet: ( A ) Representative confocal 3D images of ZO-1 (green) immunostained confluent monolayers of heterozygous-talin1 primary ECs transfected with full-length talin1 protein (EC-Tln WT ) or SCLS- TLN1 mutant lacking the 21 aa of exon 54 (EC-Tln Δex54 ) or the talin1 ABS3 mutation, R2510A (EC-Tln ABS3 ). Nuclei were stained with DAPI (blue). ( B ) Color scaling of the ZO-1 staining area, whereby the red color is the highest continuous staining area (>70 μm 2 ), decreasing to smaller areas marked by different colors until it reaches the lowest measurements, which are of blue-violet color (<30 μm 2 ), analyzed by IMARIS. Scale bars: 10 μm. ( C ) Graph displays the quantification of the continuous junctional area, as represented by the percentage of staining surfaces greater than 30 μm 2 versus the total staining area. Data represent the mean area per field of view. n fields of view analyzed: EC-Tln WT = 13; EC-Tln Δex54 = 13, EC-Tln ABS3 = 8. Red dots represent the mean ± SEM of 3 independent experiments. *** P = 0.0002, **** P < 0.0001 by 1-way ANOVA with Dunnett’s multiple-comparison test. ( D ) Graph displays the distribution of 3 different indexes of junctional fragments as a percentage of the total staining surfaces. Data represent the mean ± SEM of 3 independent experiments. n fields of view analyzed: EC-Tln WT = 14; EC-Tln Δex54 = 11, EC-Tln ABS3 = 9.

Article Snippet: Primary antibodies anti-talin1 (clone 97H6, Bio-Rad, MCA4770, 1:500), anti-talin2 (custom made, 1:50; ref. ), anti-paxillin (BD Biosciences, 610052, 1:200), anti–p-Tyr118-paxillin (Cell Signaling Technology, 2541, 1:200), anti-vinculin (Sigma-Aldrich, V9264, 1:400), anti–p-Tyr397-FAK (Biosource, 44624G, 1:100), or anti–integrin-β1 (clone 9EG7, BD Biosciences, 550531) were added on the coverslips in 0.1% BSA/PBS blocking buffer and incubated overnight at 4°C.

Techniques: Transfection, Mutagenesis, Staining, Comparison

Journal: JCI Insight

Article Title: Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

doi: 10.1172/jci.insight.173664

Figure Lengend Snippet: ( A ) Representative confocal 3D images of VE-cadherin (green) and actin (magenta) immunostained confluent monolayers of heterozygous-talin1 primary ECs transfected with full-length talin1 protein (EC-Tln WT ) or SCLS- TLN1 mutant lacking the 21 aa of exon 54 (EC-Tln Δex54 ) or the talin1 ABS3 mutation, R2510A (EC-Tln ABS3 ). Nuclei were stained with DAPI (blue). ( B ) Single actin staining of the panels in A . ( C ) 3D surfaces of actin staining generated in IMARIS software. Scale bars: 10 μm. Representative images of 3 independent experiments performed with different primary EC populations.

Article Snippet: Primary antibodies anti-talin1 (clone 97H6, Bio-Rad, MCA4770, 1:500), anti-talin2 (custom made, 1:50; ref. ), anti-paxillin (BD Biosciences, 610052, 1:200), anti–p-Tyr118-paxillin (Cell Signaling Technology, 2541, 1:200), anti-vinculin (Sigma-Aldrich, V9264, 1:400), anti–p-Tyr397-FAK (Biosource, 44624G, 1:100), or anti–integrin-β1 (clone 9EG7, BD Biosciences, 550531) were added on the coverslips in 0.1% BSA/PBS blocking buffer and incubated overnight at 4°C.

Techniques: Transfection, Mutagenesis, Staining, Generated, Software

Journal: JCI Insight

Article Title: Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

doi: 10.1172/jci.insight.173664

Figure Lengend Snippet: ( A ) Basal, ( B ) thrombin-induced, and ( C ) VEGF-induced leakage of FITC-dextran through full-length WT control talin1 (EC-Tln WT ), SCLS- TLN1 mutant (EC-Tln Δex54 ), and talin1 ABS3 R2510A mutant (EC-Tln ABS3 ) endothelial monolayers, as measured by the Transwell assay. ( A ) Scatter plots display the values of fluorescence intensity (arbitrary units) of at least 3 independent experiments. n EC-Tln WT = 4; n EC-Tln Δex54 = 4, n EC-Tln ABS3 = 3. ( B and C ) Scatter plots display the fold increase over the basal permeability in each monolayer induced by ( B ) thrombin in at least 3 independent experiments or ( C ) VEGF in 2 independent experiments. * P = 0.0136; ** P = 0.0034 ( A ); ** P = 0.0043; ** P = 0.0011 ( B ) by 1-way ANOVA with Dunnett’s multiple-comparison test. ns, no statistical significance ( C ). ( D – F ) Representative confocal 3D images of VE-cadherin (green) immunostained confluent monolayers of heterozygous-talin1 primary ECs transfected with ( D ) EC-Tln WT or ( E ) EC-Tln Δex54 or ( F ) EC-Tln ABS3 . Nuclei were stained with DAPI (blue) Scale bars: 10 μm. ( G ) Graph displays the quantification of the continuous junctional VE-cadherin area, as represented by the percentage of staining surfaces greater than 30 μm 2 versus the total staining area with and without VEGF stimulation. Data represent the mean area per monolayer. n = 6 fields of view analyzed. Red symbols represent the mean ± SEM of 2 independent experiments. * P < 0.02, ** P < 0.002 by unpaired, 2-tailed t test.

Article Snippet: Primary antibodies anti-talin1 (clone 97H6, Bio-Rad, MCA4770, 1:500), anti-talin2 (custom made, 1:50; ref. ), anti-paxillin (BD Biosciences, 610052, 1:200), anti–p-Tyr118-paxillin (Cell Signaling Technology, 2541, 1:200), anti-vinculin (Sigma-Aldrich, V9264, 1:400), anti–p-Tyr397-FAK (Biosource, 44624G, 1:100), or anti–integrin-β1 (clone 9EG7, BD Biosciences, 550531) were added on the coverslips in 0.1% BSA/PBS blocking buffer and incubated overnight at 4°C.

Techniques: Control, Mutagenesis, Transwell Assay, Fluorescence, Permeability, Comparison, Transfection, Staining

Journal: JCI Insight

Article Title: Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

doi: 10.1172/jci.insight.173664

Figure Lengend Snippet: ( A ) Representative confocal 3D images of VE-cadherin (green) and vinculin (magenta) immunostained confluent monolayers of heterozygous-talin1 ECs transfected with full-length talin 1 (EC-Tln WT ), SCLS- TLN1 mutant lacking the 21 aa of exon 54 (EC-Tln Δex54 ), or the talin1 ABS3 mutation (EC-Tln ABS3 ). Vinculin signal alone (magenta) and the colocalization signal of VE-cadherin/vinculin (white) are shown in the middle and bottom panels, respectively. Nuclei were stained with DAPI (blue). ( B ) Graph displays the quantification of the colocalization area of VE-cadherin and vinculin normalized to the number of nuclei present in each field of view. ( C ) Western blot analysis of total vinculin expression levels in control and SCLS-modeled ECs. HSC70 served as a loading control. ( D ) Graph displays the quantification of the percentage of vinculin colocalized with p-Y31-paxillin at dynamically remodeled adhesion sites. In all graphs, data represent the percentage mean colocalization area per image; n fields of view analyzed for B : EC-Tln WT = 21; EC-Tln ABS3 = 11; EC-Tln Δex54 = 11 and for D : EC-Tln WT = 14; EC-Tln ABS3 = 7; EC-Tln Δex54 = 7. Red symbols represent the mean ± SEM of 3 independent experiments for B and 2 independent experiments for D . *** P < 0.0003 ( B ) and * P = 0.0507; ** P = 0.0085 ( D ) by 1-way ANOVA with Dunnett’s multiple-comparison test. Scale bars: 10 μm.

Article Snippet: Primary antibodies anti-talin1 (clone 97H6, Bio-Rad, MCA4770, 1:500), anti-talin2 (custom made, 1:50; ref. ), anti-paxillin (BD Biosciences, 610052, 1:200), anti–p-Tyr118-paxillin (Cell Signaling Technology, 2541, 1:200), anti-vinculin (Sigma-Aldrich, V9264, 1:400), anti–p-Tyr397-FAK (Biosource, 44624G, 1:100), or anti–integrin-β1 (clone 9EG7, BD Biosciences, 550531) were added on the coverslips in 0.1% BSA/PBS blocking buffer and incubated overnight at 4°C.

Techniques: Transfection, Mutagenesis, Staining, Western Blot, Expressing, Control, Comparison

Journal: JCI Insight

Article Title: Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

doi: 10.1172/jci.insight.173664

Figure Lengend Snippet: In WT ECs, vinculin is dynamically distributed between both cell-ECM and cell-cell adhesions to regulate their dynamics. In SCLS- TLN1 mutant ECs with heterozygous disruption of talin1 R13 domain, the vinculin localization at adherens junctions is severely impaired, leading to defective endothelial barrier function. The disorganization of adherens junctions observed in the SCLS- TLN1 ECs is reproduced by a talin1 mutant with defective ABS3 binding to actin. Therefore, we propose that the SCLS- TLN1 mutant destabilizes endothelial adherens junctions by perturbing the force loading on talin. This in turn results in sequestering of vinculin at cell-ECM adhesions, depleting it from adherens junctions, which leads to defective remodeling of the cell-cell junctions.

Article Snippet: Primary antibodies anti-talin1 (clone 97H6, Bio-Rad, MCA4770, 1:500), anti-talin2 (custom made, 1:50; ref. ), anti-paxillin (BD Biosciences, 610052, 1:200), anti–p-Tyr118-paxillin (Cell Signaling Technology, 2541, 1:200), anti-vinculin (Sigma-Aldrich, V9264, 1:400), anti–p-Tyr397-FAK (Biosource, 44624G, 1:100), or anti–integrin-β1 (clone 9EG7, BD Biosciences, 550531) were added on the coverslips in 0.1% BSA/PBS blocking buffer and incubated overnight at 4°C.

Techniques: Mutagenesis, Disruption, Binding Assay