FIG 4

CEACAM 5 interacts with MERS-CoV spike. (A) Surface and intracellular expression of CEACAM5-V5 were verified by flow cytometry. (B) For co-IP, BHK21 cells were transfected with CEACAM5-V5 (lanes 1 and 2 from left) or empty vector (lane 3). The cell lysate was immunoprecipitated with MERS-CoV S1-FLAG (lanes 1 and 3) or E. coli BAP-FLAG protein (lane 2) preadsorbed onto anti-FLAG M2 agarose beads prior to SDS-PAGE. The protein complex was detected by using the anti-FLAG antibody or the anti-V5 antibody. (C) Reciprocal co-IP was performed using CEACAM5-V5 as the bait. Purified MERS-CoV S1-FLAG (lanes 1 and 3) or BAP-FLAG (lane 2) proteins were immunoprecipitated with overexpressed CEACAM5-V5 or pcDNA-V5 protein preadsorbed onto anti-V5 Sepharose beads. Western blots were detected with the anti-FLAG or the anti-CEACAM5 antibody. (D) Endogenous co-IP was performed with MERS-CoV-infected or mock-infected Huh7 cell lysates using the rabbit anti-CEACAM5 antibody, the rabbit anti-MERS-CoV spike antibody, or the rabbit isotype IgG. Western blots were detected with the rabbit anti-MERS-CoV spike antibody or the rabbit anti-CEACAM5 antibody.