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

Journal: American Journal of Physiology - Cell Physiology

Article Title: Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

doi: 10.1152/ajpcell.00199.2016

Figure Lengend Snippet: Basal phosphorylation levels of protein kinases. Total cell lysates collected from AAC-19 and LM-a3-1 cells were separated by SDS-PAGE and analyzed by Western blotting for pScr (Src pY418) (A), pERK (B), and pAkt (C). A representative Western blot is shown, and the quantitative data are means ± SE from at least three independent experiments. *P < 0.05 compared with AAC-19.

Article Snippet: The polyclonal anti-Src[pY418] antibody (raised in rabbit; Catalog No. 44660G), cell culture media, fetal bovine serum, trypsin, and Lipofectamine 2000 were from Invitrogen.

Techniques: SDS Page, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

doi: 10.1152/ajpcell.00199.2016

Figure Lengend Snippet: Effect of Na/K-ATPase peptides on Src by in vitro kinase assay. A: amino acid sequence of α1 NaKtide and α3 NaKtide among different species. The amino acids of α3 NaKtide that are different from those of α1 NaKtide are in bold and underlined. B: Src (4.5 units) was incubated with different concentrations of NaKtide and α3 NaKtide for 15 min and then assayed for pY418 Src. Quantitative data from at least three independent experiments are shown as means ± SE. *P < 0.05 compared with control.

Article Snippet: The polyclonal anti-Src[pY418] antibody (raised in rabbit; Catalog No. 44660G), cell culture media, fetal bovine serum, trypsin, and Lipofectamine 2000 were from Invitrogen.

Techniques: In Vitro, Kinase Assay, Sequencing, Incubation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase

doi: 10.1152/ajpcell.00199.2016

Figure Lengend Snippet: Effect of ouabain on cell signaling. Cells were serum starved, treated with 10 and 100 µM ouabain for 10 min, and harvested. Total cell lysates collected from AAC-19 and LM-α3-1 cells were separated by SDS-PAGE and analyzed by Western blotting for Src pY418 and c-Src (A), pERK and ERK (B), and pAkt and Akt (C). A representative Western blot is shown, and the quantitative data are means ± SE from at least three independent experiments. *P < 0.05 and **P < 0.01 compared with control.

Article Snippet: The polyclonal anti-Src[pY418] antibody (raised in rabbit; Catalog No. 44660G), cell culture media, fetal bovine serum, trypsin, and Lipofectamine 2000 were from Invitrogen.

Techniques: SDS Page, Western Blot