Fig. 6.

PLAC8 does not affect binding and internalization of SADS-CoV. Binding (A) and uptake (B) assay of SADS-CoV on Scramble and PLAC8 KO cells. For binding, cells were incubated with virus (MOI: 0.5) at 4 °C for 1 h and washed three times with HBSS. For uptake, after the wash from the binding assay, cells were incubated at 37 °C for 1 h. Surface virions were removed by 1) trypsin wash, 2) pH 2.5 acid wash, and 3) HBSS wash. Total RNA was isolated, and viral gRNA was determined by qRT-PCR. Data were plotted in a box-and-whisker plot, with the boxes representing 95% CI and whiskers representing maximum and minimum values from four independent experiments with three replicates. (C) Immunoprecipitation of PLAC8 with SADS-CoV spike protein. hPLAC8-FLAG and SADS-CoV spike with a C-terminal C9 tag were cotransfected in 293T cells for 48 h. Magnetic beads against FLAG tag were used for IP and blotted with α-PLAC8 or α-C9 antibodies. PLSCR1, a known factor that interacts with PLAC8, was used as the positive control, and PLAC8 without a FLAG tag was used as the negative control. I, input; FT, flow through; B, bound; ns, not significant; IP, immunoprecipitation; IB, immunoblot.