FIG 2.
Interactions between ITGB1 and RABV G. (A) ITGB1 interacted with RABV G. (I) Plasmid ITGB1-Flag and ERAG-Myc were cotransfected in HEK293 cells at 37°C for 48 h, and ITGB1-Flag interacted with ERAG-Myc in co-IP assays with HEK293 cell lysates. (II) HEK293 cells were infected with a recombinant ERA fused with a Myc tag at the C terminus of glycoprotein (rERAG/Myc) at an MOI of 0.1 for 36 h after transfection with plasmid ITGB1-Flag for 12 h, and ITGB1-Flag interacted with ERAG-Myc from rERAG/Myc in co-IP assays. (III) HEK293 cells were infected with a recombinant ERA fused with a Flag tag at the C terminus of glycoprotein (rERAG/Flag) at an MOI of 0.1 for 48 h, and endogenous ITGB1 interacted with ERAG-Flag from rERAG/Flag in co-IP assays. (IV) Myc-tagged RABV G plasmids from mouse-adapted strain CVS-24 (CVS24G-Myc), street virus strain GX/09 (GX/09G-Myc), or west Caucasian bat virus (WCBVG-Myc) were cotransfected with plasmid ITGB1-Flag in HEK293 cells for 48 h, and the indicated RABV G interacted with ITGB1-Flag in co-IP assays. (B) ITGB1 ectodomain (aa 1 to 728, ITGB1ED-Flag) interacted with ERA G ectodomain (aa 20 to 459, ERAGED-Myc). (I) ITGB1-Flag interacted with ERAGED-Myc but not ERA G transmembrane and cytoplasmic domain (aa 460 to 524, ERAGTC-Myc) in co-IP assays with plasmid-cotransfected HEK293 cell lysates. (II) ITGB1ED-Flag but not ITGB1 transmembrane and cytoplasmic domain (aa 729 to 798, ITGB1TC-Flag) interacted with ERAGED-Myc in co-IP assays with plasmid-cotransfected HEK293 cell lysates. (C) Purified GST-tagged ITGB1 ectodomain (aa 21 to 728, ITGB1ED), but not GST-tagged irrelative protein (IRP), pulled down purified His-tagged ERAG ectodomain (aa 41 to 450, ERAGED) using pulldown assay.