Goat serum

Hela cells were transfected the indicated plasmids, and SLK.iBAC cells were induced with Dox (1 μg/mL) for 24h. Then the cells were washed three times with PBS and fixed with 4% PFA for 20 min. Subsequently, the cells were permeabilized with 1% Triton X-100 for 10 min and blocked with 10% goat serum (Antgene, Wuhan, China) for 1 h at room temperature. Next, Hela cells were incubated with rabbit anti-GM130 polyclonal antibody (1:200; A11408; ABclonal) or mouse anti-calnexin monoclonal antibody (1:100; sc-23954; Santa Cruz), while SLK.iBAC cells were incubated with rabbit anti-pORF55 polyclonal antibody (1:200) and mouse anti-GM130 monoclonal antibody (1:200; AB-398142; BD Bioscience) overnight at 4°C.Then the cells were washed with PBST (Phosphate Buffered Saline with 0.05% Tween-20, pH 7.4) for three times, and were incubated with the secondary antibodies for 90 min at room temperature. After washing with PBST, the slides were mounted with DAPI Fluoromount-G mounting medium (SouthernBiotech). The images were collected with a confocal microscope (Zeiss LSM880), and the pictures were processed using ZEN 2 (Zeiss) and ImageJ (NIH).

SLK.iBAC-GFP cells were induced with Dox (1 μg/ml) for 24 h to trigger lytic reactivation. The cells were then washed three times with PBS and fixed with 4% (w/v) paraformaldehyde (PFA) (#DF0131, LEAGENE, Beijing, China) for 10 min. Next, the fixed cells were washed with PBS for three times, permeabilized with 1% Triton X-100 for 5 min, and blocked with 10% goat serum (Antgene, Wuhan, China) for 1 hour at room temperature. After washing with wash buffer (1X PBS with 0.1% Tween 20) for three times, the cells were incubated with rabbit anti-K-RTA or mouse anti-FLAG antibodies diluted in PBS containing 1% BSA at 4°C overnight. Cells were washed with wash buffer (PBS containing 0.05% Tween-20) and then incubated with Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (1:1000; Invitrogen) and Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (1:1000; Invitrogen) at room temperature for 1 hour. After washing with wash buffer and ultrapure water, the slides were mounted with DAPI Fluoromount-G mounting medium (SouthernBiotech). Finally, the images were acquired with a laser scanning confocal microscopy (Leica Stellaris 5) and processed using Image J and Leica image browser [60 (link)].