Figure S2.
Cortactin is not degraded by TNFα stimulation or endothelial enzymes. (A) Immunoblot of cortactin, ICAM-1, and GAPDH in lysates of HUVEC monolayers stimulated with TNFα (15 ng/ml) for the indicated times. The increase in ICAM-1 shows that the inflammatory stimulus worked. Quantification of cortactin pixel intensities normalized to tubulin expression and unstimulated controls (set to 1, dotted line) is shown in the graph on the right (n = 3). (B) Representative flow cytometry dot plots of TNFα-treated HUVEC alone (left, cortactin+CD45−) and co-cultures with human neutrophils (right, cortactin− CD45+) for 20 min (C–F) HUVEC monolayers were treated with the proteasome inhibitor MG-132 and the ERK inhibitor PD98059 (C and D), the calpain inhibitor ALLN (E), the lysosome inhibitor leupeptin (F), or DMSO as vehicle control for 2 h before co-culture with neutrophils for 20 min. Cortactin protein levels were then analyzed by flow cytometry and displayed as MFIs; n = 2–4 independent experiments. ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. Source data are available for this figure: SourceData FS2. Refer to the image caption for details. Panel A shows an immunoblot with bands for ICAM-1, cortactin, and GAPDH in HUVEC monolayers stimulated with TNF for different times. The graph on the right displays the relative pixel intensity of cortactin normalized to tubulin expression and unstimulated controls. Panel B presents two flow cytometry dot plots showing cortactin and CD45 levels in TNF-treated HUVEC alone and co-cultured with human neutrophils. Panel C is a bar graph showing endothelial cortactin mean fluorescence intensity (MFI) after treatment with proteasome inhibitor MG-132 and ERK inhibitor PD98059. Panel D is a bar graph showing endothelial cortactin MFI after treatment with varying concentrations of MG132. Panel E is a bar graph showing endothelial cortactin MFI after treatment with varying concentrations of ALLN. Panel F is a bar graph showing endothelial cortactin MFI after treatment with leupeptin. Each graph includes error bars and statistical significance indicators.

Cortactin is not degraded by TNFα stimulation or endothelial enzymes. (A) Immunoblot of cortactin, ICAM-1, and GAPDH in lysates of HUVEC monolayers stimulated with TNFα (15 ng/ml) for the indicated times. The increase in ICAM-1 shows that the inflammatory stimulus worked. Quantification of cortactin pixel intensities normalized to tubulin expression and unstimulated controls (set to 1, dotted line) is shown in the graph on the right (n = 3). (B) Representative flow cytometry dot plots of TNFα-treated HUVEC alone (left, cortactin+CD45−) and co-cultures with human neutrophils (right, cortactin− CD45+) for 20 min (C–F) HUVEC monolayers were treated with the proteasome inhibitor MG-132 and the ERK inhibitor PD98059 (C and D), the calpain inhibitor ALLN (E), the lysosome inhibitor leupeptin (F), or DMSO as vehicle control for 2 h before co-culture with neutrophils for 20 min. Cortactin protein levels were then analyzed by flow cytometry and displayed as MFIs; n = 2–4 independent experiments. ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. Source data are available for this figure: SourceData FS2.

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