Pla assay

RD cells were infected with EV71 (MOI = 5) for 6 h, fixed with 4% PFA for 20 min, washed three times with 1×PBS, and then permeabilized with Triton X-100 for 20 min. The PLA assay was performed as previously described (Yu et al., 2023 (link)). In brief, cells were blocked at room temperature for 1 h with blocking solution (DUO82007) and then incubated with a primary antibody in dilution buffer (mouse anti-3D antibody: 1:100; rabbit anti-RagB: 1:75) overnight at 4°C. Next, the cells were subjected to the PLA assay (Cat #DUO92008; Sigma-Aldrich) with anti-mouse (Cat #DUO92001, RRID: AB_2810939; Sigma-Aldrich) and anti-rabbit (Cat #DUO92005, RRID: AB_2810942; Sigma-Aldrich) secondary antibodies following the manufacturer’s instructions. Nuclei were stained with DAPI. Confocal images were taken using a Leica TCS SP8 MP.

RD cells were infected with EV71 (MOI = 5) for 6 h, fixed with 4% PFA for 20 min, washed three times with 1×PBS, and then permeabilized with Triton X-100 for 20 min. The PLA assay was performed as previously described (Yu et al., 2023 (link)). In brief, cells were blocked at room temperature for 1 h with blocking solution (DUO82007) and then incubated with a primary antibody in dilution buffer (mouse anti-3D antibody: 1:100; rabbit anti-RagB: 1:75) overnight at 4°C. Next, the cells were subjected to the PLA assay (Cat #DUO92008; Sigma-Aldrich) with anti-mouse (Cat #DUO92001, RRID: AB_2810939; Sigma-Aldrich) and anti-rabbit (Cat #DUO92005, RRID: AB_2810942; Sigma-Aldrich) secondary antibodies following the manufacturer’s instructions. Nuclei were stained with DAPI. Confocal images were taken using a Leica TCS SP8 MP.

Cells grown on coverslips were incubated on ice with anti-IFNAR1 or anti-IFNAR2 antibody for 40 min and washed twice with ice-cold PBS. Cells were stimulated with IFN-α at 37 °C for the indicated times. Fixation and permeabilization were performed as described above. Cells were incubated with primary antibodies as indicated and processed for the PLA assay following the manufacturer's protocol (Sigma-Aldrich). Briefly, cells were incubated with secondary antibodies conjugated to oligonucleotide primers (labelled as probe PLUS and probe MINUS) for 1 h at 37 °C. Ligase was added and the two olignucleotides were ligated provided that they were in close proximity (<40 nm). In the last stage—rolling circle amplification—polymerase and fluorescently labelled nucleotides were used to create a fluorescent reaction product reflecting protein–protein interactions. Reaction products were visible as fluorescent dots and imaged using epifluorescence inverted microscope Leica DM 6000B equipped with a HCX PL Apo 633, NA 1.40, oil-immersion objective and an electron-multiplying CCD camera (Photometrics CoolSNAP HQ). Fluorescent dots were quantified using ImageJ and ‘find maxima' option with a noise tolerance parameter settled visually. Total numbers of dots on images were normalized to the total numbers of cells visualized by 4,6-diamidino-2-phenylindole staining.