Normal goat serum (ngs)

The day before, HEK293T cells stably expressing the EBNA1-FLAG-HA protein were seeded at a density of 5 × 104 cells/well in 24-well plates. Cells were washed twice with PBS and fixed 20 min using 4% paraformaldehyde and 4% sucrose in PBS at room temperature. Cells were then permeabilized with 0.15% triton X-100 in PBS for 5 min at room temperature and blocked in 10% normal goat serum (Wisent). Anti-HA antibody (Santa Cruz Biotechnologies) was incubated 4 h at room temperature to allow detection of EBNA1-FLAG-HA protein. Cells were washed and incubated 1 h in the dark with DyLight 488-labelled goat anti-mouse secondary antibody (ThermoFisher Scientific). Nucleus staining was performed using 1 μg/ml Hoechst, 15 min at room temperature. Cover glasses were mounted on slides with SlowFadeGold mounting medium (Life Technologies), then epifluorescence microscopy was conducted using a Nikon Eclipse TE2000-E visible/epifluorescence inverted microscope using bandpass filters for Hoechst and DyLight 488.

Immunofluorescence and confocal analyses were carried out as previously described with slight modifications (3 (link)). Briefly, HeLa cells were fixed for 20 min with 4% paraformaldehyde (PFA), then incubated for 30 min in 10% normal goat serum (Wisent, #053-150) and 0.1% saponin (Sigma) in PBS. Cells were incubated in the same buffer with anti-Flag (1/1000) and anti-HA (1/500, chicken) primary antibodies and with anti-mouse-Alexa Fluor488 (1/1000) and anti-chicken-Atto594 (1/500) or anti-mouse-Alexa Fluor647 (1/1000) and anti-chicken-Atto390 (1/100) secondary antibodies. Cells were then examined with a scanning confocal microscope (FV1000; Olympus) coupled to an inverted microscope with a 63x oil-immersion objective (Olympus).
For live-cell staining, HeLa cells grown on coverslips were washed with DMEM and incubated for 30 min on ice with anti-Flag (1/1000) and anti-HA (1/500, chicken) in DMEM. After washing, cells were incubated for 30 min on ice with anti-mouse-Alexa Fluor488 (1/1000) and anti-chicken-Atto594 (1/500) antibodies diluted in DMEM. After washing, cells were processed as described above.

Following cell adhesion, coverslips were washed twice with PBS and fixed with 4% paraformaldehyde pH 7.4 (BioShop) for 15 minutes at room temperature. After three washes with PBS, fixed cells were permeabilized using 0.2% Triton X-100 (Sigma-Aldrich) for 15 minutes at room temperature. Coverslips were blocked in PBS supplemented with 10% normal goat serum (Wisent) and 0.1% NP40 (Sigma-Aldrich) for 1 hour at room temperature and incubated with the following antibodies diluted in blocking solution: mouse FLAG (Sigma-Aldrich), mouse GRB2 (BD Biosciences), rabbit GRB2 (Santa Cruz) or rabbit MPZL1 (Cell Signalling Technology) for 1 hour at room temperature and then overnight at 4 °C. After washes in 0.1% NP40 in PBS, coverslips were incubated with Alexa 568-conjugated goat anti-rabbit (Invitrogen) or Alexa 488-conjugated goat anti-mouse (Cell Signalling Technology) antibodies for 1 hour at room temperature. They were washed three times with 0.1% NP40 in PBS and twice with PBS before being mounted on slides using ProLong Gold antifade with DAPI (Thermo Fisher). Pictures were acquired with an Olympus FV1000 using the FluoView software or with a Nikon Eclipse E600 imaging system using MetaView.