Metavue 4

The proximity ligation assay (PLA) was performed using a Duolink In Situ Red Starter Kit Mouse/Rabbit available from Millipore Sigma (Cat no. DUO92101) following manufacturer’s instructions. Briefly, after blocking, cells were incubated with rabbit polyclonal SOCS1 (Abcam, ab137384) and mouse monoclonal α-tubulin (Sigma, T6199) antibodies. After washing, incubation with mixture of secondary antibodies conjugated with oligonucleotides (anti-rabbit PLUS and anti-mouse MINUS PLA probes) was done and proximity ligation reaction was continued using Duolink In Situ Detection reagents. Red PLA signals were visualized, and images were captured in EVOS fluorescence microscope (ThermoFisher Scientific). Reaction without antibodies served as a negative control. PLA signals (red fluorescence) were analyzed using MetaVue 4.6 (Universal Imaging, Downington, PA). The values were statistically processed using Sigma Plot 7.1 (SPSS Science, Chicago, IL) software. For each experimental condition, at least 10 microscopic fields in each independent experiment were analyzed. To demonstrate co-localization of PLA signal with the MT, immunofluorescence staining of MT with β-tubulin antibodies was performed after the PLA assay.

Confluent EC grown on coverslips were exposed to experimental conditions, fixed with 3.7% formaldehyde, and permeabilized with 0.25% Triton X-100. After blocking with 2% bovine serum albumin, cells were exposed to primary antibodies for 60 min. Fluorescently-tagged secondary antibodies were applied for 60 min. Cells were imaged using a Nikon video imaging system. To quantify Texas Red stained actin stress fibers, images were analyzed with respect to the ratio of stress fiber area relative to whole cells using MetaVue 4.6 (Universal Imaging, Downington, PA) and statistically processed using Sigma Plot 7.1 (SPSS Science, Chicago).