Human Umbilical Vein Endothelial Cells (HUVEC) were grown to 80% confluence on gelatin-coated glass coverslips, then infected or transfected as indicated. After 72 hours, cells were fixed with 3.7% formaldehyde in Phosphate-Buffered-Saline (PBS) for 10 minutes (RT) and subsequently permeabilized with 0.1% Triton X-100 in PBS for 5 minutes (RT). Coverslips were then blocked for 1 hour (37°C) using 10% normal goat serum in PBS. Immunostainings were performed with the indicated antibodies (60 min; RT). Imaging was performed with a Perkin Elmer UltraView Vox Spinning Disk Confocal using a 40x/NA-1.30 or a 60x/NA-1.42 oil objective.
For the calcium switch assay, HUVEC were grown to 80% confluence on gelatin-coated glass coverslips, then infected/transfected as indicated. 72 Hours after infection cells were washed with cold PBS and then incubated with DMEM (5% FBS; 4 mM EGTA) for 30 minutes at 37°C. Cells were then washed with cold PBS and incubated with DMEM (2 mM CaCl; 100 µM 8-pCPT-2’-O-Me-cAmp) for 20 minutes at 37°C. Next, cells were stained as described above. When indicated, to inhibit Rho Kinase activity cells received ROCK inhibitor H-1152 (3 µM) for 30 minutes (37°C) prior to calcium switch assay.
For the calcium switch assay, HUVEC were grown to 80% confluence on gelatin-coated glass coverslips, then infected/transfected as indicated. 72 Hours after infection cells were washed with cold PBS and then incubated with DMEM (5% FBS; 4 mM EGTA) for 30 minutes at 37°C. Cells were then washed with cold PBS and incubated with DMEM (2 mM CaCl; 100 µM 8-pCPT-2’-O-Me-cAmp) for 20 minutes at 37°C. Next, cells were stained as described above. When indicated, to inhibit Rho Kinase activity cells received ROCK inhibitor H-1152 (3 µM) for 30 minutes (37°C) prior to calcium switch assay.