293T cell monolayers were transfected in T25 flasks with the indicated plasmids using FuGENE 6 (Promega). Transfected cells were incubated for 72 hours in a 37°C incubator with 5% CO2. Cells were detached with gentle tapping, were pelleted by centrifugation at 750g, and resuspended in 200 μl of fluorescence-activated cell sorting (FACS) buffer (PBS and 5% FBS). Cells were incubated (1:100 dilution) with mAbs and incubated for 1 hour at room temperature. Cells were then pelleted by centrifugation at 750g and washed three times with FACS buffer. Cells were then incubated with goat anti-mouse Alexa Fluor 488–conjugated secondary antibody (1:500; Invitrogen) for 30 min at room temperature, washed three times, and resuspended in 1 ml of FACS buffer. Flow cytometry was performed on a FACSCalibur flow cytometer (Becton Dickinson). Data were collected and analyzed using FlowJo software (Tree Star Inc., Ashland, OR). A total of 10,000 cells were analyzed for each sample using a live gate.

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