Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.; Buchmeier, Michael J.

Flow cytometry. Transfected HEK 293T cells were collected and disassociated using cold 5 mM EDTA in 1ϫ PBS. Cells were resuspended in 5% fetal bovine serum (FBS), passed through a 26-gauge syringe, and blocked on ice for 30 min. Cells were incubated with either the anti-HA antibody (Sigma), the GP1 antibody directly conjugated to Alexa Fluor 488 (2/11/10-488), or the normal mouse IgG antibody (BioLegend) for 30 min on ice. Cells were pelleted (100 ϫ g, 3 min, 10°C) and extensively washed with 5% FBS. For the HA antibody or normal IgG samples, cells were stained with the Alexa Fluor 488 secondary antibody for 30 min on ice. Cells were washed twice with 5% FBS prior to one wash with 1ϫ PBS. Cells were transferred to prechilled fluorescence-activated cell sorting (FACS) tubes prior to 7-AAD viability staining (BioLegend). Flow cytometry was performed using the FACSCalibur and CellQuest Pro software (BD Biosciences). All data were analyzed using FlowJo software (Ashland, OR).