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. 2017 Feb 14;8(1):e00031-17. doi: 10.1128/mBio.00031-17

FIG 3 .

FIG 3 

Identification of hJAM1 protein as a binding partner for Hom-1 particles. (A) Recombinant baculovirus expressing Hom-1 VP1 was constructed by recombining a transfer vector (pFastBac83/2) carrying VP1 and VP2 sequences into a bacmid by using Bac-to-Bac technology (Thermo Fischer Scientific, Inc.). An ATG codon was inserted at the beginning of the predicted VP1 gene during PCR cloning of the Hom-1 sequence. (B) Comparison of Hom-1 VP1 synthesized in Vero cells and in insect cells. Vero cells infected with Hom-1 at an MOI of 5 were collected at 8 hpi. After the cells were lysed, the lysate proteins were resolved by SDS-PAGE in a 4 to 20% polyacrylamide gel (Thermo Fischer Scientific, Inc.) and transferred onto a nitrocellulose membrane. The protein (0.6 µg) prepared from VLPs purified with a CsCl gradient from Sf9 cells infected with Hom-1 VP1/VP2 baculovirus was analyzed along with lysates of the infected Vero cells. The membrane was probed with anti-810VLP serum. (C) EM of negatively stained Hom-1 VLPs. The scale bar corresponds to 100 nm. (D) Schematic of VLP-binding screening of the hPMP expression library. Expression vectors encoding 3,559 full-length hPMPs and ZsGreen were arrayed on microarray slides and used for reverse transfection of human cells. Cells expressing hPMPs were treated with VLPs, and bound VLPs were detected with guinea pig anti-VLP serum and fluorescent anti-guinea pig antibodies.