Cells on an eight-well Lab-Tek II chamber (Nunc) were fixed with 4% formaldehyde in PBS at RT for 10 min, permeabilized with 0.1% Triton X-100 in PBS, blocked with 1% BSA in PBS at RT for 1 hour, and incubated with primary antibodies at RT for 1 hour. Primary antibodies for FOXO1 (Cell Signaling) and TIE2 (R&D Systems) were used. The cells were then incubated with secondary antibodies (Invitrogen) in the dark at RT for 1 hour and mounted with VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (Vector Labs). Images were taken with a confocal laser scanning microscope (LSM880, Carl Zeiss).

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