Journal: Inflammation

Article Title: Focal Adhesion Kinase Activity and Localization is Critical for TNF-α-Induced Nuclear Factor-κB Activation

doi: 10.1007/s10753-020-01408-5

Figure Lengend Snippet: Antibodies Used in This Study

Article Snippet: pY418 Src , Cell Signal Technology, 2101 , Rabbit , 1:2000 (WB) , AB_331697.

Techniques: Recombinant

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet: ( A ) Representative maximum intensity projections of similar sized tufts from Nos3 +/+ and Nos3 S1176A/S1176A retinas showing VE-cadherin (green), pY685 VE-cadherin (red), and isolectin B4 (IB4; cyan). Scale bar = 50 μm. ( B ) Ratio of pY685 positive area/total VE-cadherin positive area. Mean ± S.E.M n = 3–6 images per group from 5 ( Nos3 +/+ ) and 5 ( Nos3 S1176A/S1176A ) mice, three independent experiments, *p<0.05; t-test. ( C ) Representative images of VE-cadherin staining (green) and proximity ligation assay (PLA) to detect c-Src pY418 (magenta) in isolated mouse lung endothelial cells (iEC) from Nos3 +/+ and Nos3 S1176A/S1176A mice. Scale bar = 20 µM. ( D ) c-Src pY418 PLA dots detected in PBS and VEGFA (50 ng/mL)-treated iECs from Nos3 +/+ and Nos3 S1176A/S1176A mouse lungs. Data expressed as the number of dots per field of view. ( E ) c-Src pY418 PLA dots co-localized with VE-cadherin (green), normalized against total VE-cadherin area in the field of view. Mean ± S.E.M. Cells isolated from n = 4 ( Nos3 +/+ ) and 4 ( Nos3 S1176A/S1176A ) mice, from three separate experiments. *, **p<0.05, 0.01; two-way ANOVA, Sidak’s multiple comparisons test. Figure 2—source data 1. Excel file containing numerical values collected from biochemical analyses shown in , – .

Article Snippet: For immunoblotting, the following antibodies were used as primaries: mouse-anti-α−tubulin (1:1000, Sigma, T9026), mouse anti-eNOS (1:1000, Abcam, ab76198), mouse anti-eNOS pS1177 (1:1000, BD, 612392), rabbit anti-Akt (1:1000, Cell Signaling, 9272S), rabbit anti-Akt pS473 (1:1000, Cell Signaling, 4058S), rabbit anti-Src (GD11 clone) antibody (1:1000, Merck Millipore, Mouse, 05–184), rabbit anti-Src pY418 antibody (1:1000, Invitrogen, 44–660G), goat anti-VE-cadherin (1:1000, R and D, AF1002), and rabbit anti-VE-cadherin pY685 (1:1000, DOI:10.1038/ncomms2199).

Techniques: Staining, Proximity Ligation Assay, Isolation

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet: ( A ) Representative immunoblots and the effect of VEGFA (50 ng/mL; 1, 5, 10, 30 min) on eNOS, eNOS pS1177, AKT, AKT pS473, VE-cadherin, VE-cadherin pY685, c-Src, SFK pY418, and tubulin in cultured Human Retinal Microvascular Endothelial Cells (HRMEC) treated with a PBS vehicle or 1 mM L-NMMA for 1 hr. ( B ) Quantification of eNOS pS1177/total eNOS (normalized to tubulin). ( C ) Quantification of AKT pS473/total AKT (normalized to tubulin). ( D ) Quantification of VE-cadherin pY685/total VE-cadherin (normalized to tubulin). ( E ) Quantification of SFK pY418/total c-Src (normalized to tubulin) Mean ± S.E.M. n = 3 independent experiments. *, ***p<0.05, 0.001 (indicates significance between vehicle and L-NMMA-treated samples, stimulated with VEGFA as indicated). Two-way ANOVA, Sidak’s multiple comparison test.

Article Snippet: For immunoblotting, the following antibodies were used as primaries: mouse-anti-α−tubulin (1:1000, Sigma, T9026), mouse anti-eNOS (1:1000, Abcam, ab76198), mouse anti-eNOS pS1177 (1:1000, BD, 612392), rabbit anti-Akt (1:1000, Cell Signaling, 9272S), rabbit anti-Akt pS473 (1:1000, Cell Signaling, 4058S), rabbit anti-Src (GD11 clone) antibody (1:1000, Merck Millipore, Mouse, 05–184), rabbit anti-Src pY418 antibody (1:1000, Invitrogen, 44–660G), goat anti-VE-cadherin (1:1000, R and D, AF1002), and rabbit anti-VE-cadherin pY685 (1:1000, DOI:10.1038/ncomms2199).

Techniques: Western Blot, Cell Culture

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet: ( A ) Representative maximum intensity projections of tufts from Nos3 +/+ and Nos3 S1176A/S1176A retinas immunostained for VE-cadherin (green), c-Src pY418 (red), and IB4 (cyan). ( B ) Quantification of pY418-positive area/total VE-cadherin area. n = 3 images per group from 3 ( Nos3 +/+ ) and 4 ( Nos3 S1176A/S1176A ) mice from two separate experiments; t-test. ( C ) Representative images of VE-cadherin staining (green) and negative controls for proximity ligation in isolated mouse lung endothelial cells (iEC) from Nos3 +/+ mice. Controls were performed by incubation with only one of the necessary antibodies or, one of the PLUS or MINUS PLA probes (mouse and rabbit secondary antibodies), which should inhibit rolling circle DNA synthesis. This allows for the detection of non-specific ligation or uncontrolled rolling circle DNA synthesis. Scale bar = 20 µm.

Article Snippet: For immunoblotting, the following antibodies were used as primaries: mouse-anti-α−tubulin (1:1000, Sigma, T9026), mouse anti-eNOS (1:1000, Abcam, ab76198), mouse anti-eNOS pS1177 (1:1000, BD, 612392), rabbit anti-Akt (1:1000, Cell Signaling, 9272S), rabbit anti-Akt pS473 (1:1000, Cell Signaling, 4058S), rabbit anti-Src (GD11 clone) antibody (1:1000, Merck Millipore, Mouse, 05–184), rabbit anti-Src pY418 antibody (1:1000, Invitrogen, 44–660G), goat anti-VE-cadherin (1:1000, R and D, AF1002), and rabbit anti-VE-cadherin pY685 (1:1000, DOI:10.1038/ncomms2199).

Techniques: Staining, Ligation, Isolation, Incubation, DNA Synthesis

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet:

Article Snippet: For immunoblotting, the following antibodies were used as primaries: mouse-anti-α−tubulin (1:1000, Sigma, T9026), mouse anti-eNOS (1:1000, Abcam, ab76198), mouse anti-eNOS pS1177 (1:1000, BD, 612392), rabbit anti-Akt (1:1000, Cell Signaling, 9272S), rabbit anti-Akt pS473 (1:1000, Cell Signaling, 4058S), rabbit anti-Src (GD11 clone) antibody (1:1000, Merck Millipore, Mouse, 05–184), rabbit anti-Src pY418 antibody (1:1000, Invitrogen, 44–660G), goat anti-VE-cadherin (1:1000, R and D, AF1002), and rabbit anti-VE-cadherin pY685 (1:1000, DOI:10.1038/ncomms2199).

Techniques: Griess Assay, Nitrate Nitrite Colorimetric Assay, Western Blot, Sequencing, Recombinant, In Situ, Plasmid Preparation, Software

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet: ( A ) Representative maximum intensity projections of similar sized tufts from Nos3 +/+ and Nos3 S1176A/S1176A retinas showing VE-cadherin (green), pY685 VE-cadherin (red), and isolectin B4 (IB4; cyan). Scale bar = 50 μm. ( B ) Ratio of pY685 positive area/total VE-cadherin positive area. Mean ± S.E.M n = 3–6 images per group from 5 ( Nos3 +/+ ) and 5 ( Nos3 S1176A/S1176A ) mice, three independent experiments, *p<0.05; t-test. ( C ) Representative images of VE-cadherin staining (green) and proximity ligation assay (PLA) to detect c-Src pY418 (magenta) in isolated mouse lung endothelial cells (iEC) from Nos3 +/+ and Nos3 S1176A/S1176A mice. Scale bar = 20 µM. ( D ) c-Src pY418 PLA dots detected in PBS and VEGFA (50 ng/mL)-treated iECs from Nos3 +/+ and Nos3 S1176A/S1176A mouse lungs. Data expressed as the number of dots per field of view. ( E ) c-Src pY418 PLA dots co-localized with VE-cadherin (green), normalized against total VE-cadherin area in the field of view. Mean ± S.E.M. Cells isolated from n = 4 ( Nos3 +/+ ) and 4 ( Nos3 S1176A/S1176A ) mice, from three separate experiments. *, **p<0.05, 0.01; two-way ANOVA, Sidak’s multiple comparisons test. Figure 2—source data 1. Excel file containing numerical values collected from biochemical analyses shown in , – .

Article Snippet: Antibody , Anti-c-Src pY418 (rabbit polyclonal) , Invitrogen , Cat# 44–660G RRID: AB_1500523 , IF (1:100), WB (1:1000).

Techniques: Staining, Proximity Ligation Assay, Isolation

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet: ( A ) Representative immunoblots and the effect of VEGFA (50 ng/mL; 1, 5, 10, 30 min) on eNOS, eNOS pS1177, AKT, AKT pS473, VE-cadherin, VE-cadherin pY685, c-Src, SFK pY418, and tubulin in cultured Human Retinal Microvascular Endothelial Cells (HRMEC) treated with a PBS vehicle or 1 mM L-NMMA for 1 hr. ( B ) Quantification of eNOS pS1177/total eNOS (normalized to tubulin). ( C ) Quantification of AKT pS473/total AKT (normalized to tubulin). ( D ) Quantification of VE-cadherin pY685/total VE-cadherin (normalized to tubulin). ( E ) Quantification of SFK pY418/total c-Src (normalized to tubulin) Mean ± S.E.M. n = 3 independent experiments. *, ***p<0.05, 0.001 (indicates significance between vehicle and L-NMMA-treated samples, stimulated with VEGFA as indicated). Two-way ANOVA, Sidak’s multiple comparison test.

Article Snippet: Antibody , Anti-c-Src pY418 (rabbit polyclonal) , Invitrogen , Cat# 44–660G RRID: AB_1500523 , IF (1:100), WB (1:1000).

Techniques: Western Blot, Cell Culture

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet: ( A ) Representative maximum intensity projections of tufts from Nos3 +/+ and Nos3 S1176A/S1176A retinas immunostained for VE-cadherin (green), c-Src pY418 (red), and IB4 (cyan). ( B ) Quantification of pY418-positive area/total VE-cadherin area. n = 3 images per group from 3 ( Nos3 +/+ ) and 4 ( Nos3 S1176A/S1176A ) mice from two separate experiments; t-test. ( C ) Representative images of VE-cadherin staining (green) and negative controls for proximity ligation in isolated mouse lung endothelial cells (iEC) from Nos3 +/+ mice. Controls were performed by incubation with only one of the necessary antibodies or, one of the PLUS or MINUS PLA probes (mouse and rabbit secondary antibodies), which should inhibit rolling circle DNA synthesis. This allows for the detection of non-specific ligation or uncontrolled rolling circle DNA synthesis. Scale bar = 20 µm.

Article Snippet: Antibody , Anti-c-Src pY418 (rabbit polyclonal) , Invitrogen , Cat# 44–660G RRID: AB_1500523 , IF (1:100), WB (1:1000).

Techniques: Staining, Ligation, Isolation, Incubation, DNA Synthesis

Journal: eLife

Article Title: eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin

doi: 10.7554/eLife.64944

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-c-Src pY418 (rabbit polyclonal) , Invitrogen , Cat# 44–660G RRID: AB_1500523 , IF (1:100), WB (1:1000).

Techniques: Griess Assay, Nitrate Nitrite Colorimetric Assay, Western Blot, Sequencing, Recombinant, In Situ, Plasmid Preparation, Software