Immunofluorescence Staining of Infected Cells

Infected cells grown on glass coverslips were fixed using methanol at -20°C for 20 min or 3–4% formaldehyde at room temperature (RT) for 15 min, as stated in the text. Methanol-fixed samples were washed three times for 5 min with PBS and blocked using 3% BSA in PBS for 1h at RT. Formaldehyde-fixed samples were rinsed once with PBS, permeabilized with 0.2% Triton-X 100 (TTX-100) for 20 min, and then blocked as described above. MYR1(N) protein was detected with mouse anti-MYR1 antibodies [27 (link)], while MYR1(C) protein was detected with rat anti-HA antibodies or mouse anti-MYR1 primary antibodies. GRA7 protein was detected using rabbit anti-GRA7 antibodies [34 (link)]. GRA16-HA (and other HA-tagged proteins) was detected using rat anti-HA antibodies (Roche) while GRA24-Myc was detected using rabbit anti-Myc tag antibodies (Cell Signaling Technologies). Primary antibodies were detected with goat polyclonal Alexa Fluor-conjugated secondary antibodies (Invitrogen). Vectashield with DAPI stain (Vector Laboratories) was used to mount the coverslips on slides. Fluorescence was detected using a LSM710 inverted confocal microscope (Zeiss) or epifluorescence microscope, as stated in the text. Images were analyzed using ImageJ. All images shown for any given condition/staining in any given comparison/dataset were obtained using identical parameters.