Fig. 4

LRPAP1 binds with IFNAR1 and triggers IFNAR1 degradation. a Immunoblots of cytosol and membrane fraction in HEK-293T cells treated with 200 nM rLRPAP1 from 1 min to 10 min. b Immunofluorescence of RD cells infected with or without EV71 (MOI = 10) for 6 h. Cells were double stained with anti-IFNAR1 (Red) and anti-LRPAP1 (Green) (scale bar = 5 μm). c, d The C57BL/6 mouse cerebrum was separated into two hemispheres and immediately cultured in oxygen supplied ACSF with or without rLRPAP1 (200 nM) at 0 °C for 1 h (c). The cytosol and membrane fraction from brain tissue was then analysed by immunoblotting (d). e The flow cytometry analysis of cell surface IFNAR1 protein level on 4T1 cells after the treatment with or without rLRPAP1. f Immunoprecipitates (indicated antibodies) in HEK-293T cells ectopic-expressing LRPAP1 and IFNAR1 were analysed by immunoblotting. g The pull-down assay between the extracellular domain of IFNAR1 (ECD, Sino biological 13222-H08H) and lysates in HEK-293T cells overexpressing LRPAP1 were analysed by immunoblotting. h Microscale thermophoresis (MST) results for the binding affinity between the region of LRPAP1 (RAPD1P1, RAPD1P2) and ECD-IFNAR1. i Immunoblots of LRPAP1 ectopic-expressing HEK-293T cells treated with or without 20 mM MA for 4 h. j Immunoprecipitates (indicated antibodies) in HEK-293T cells ectopic-expressing ubiquitin, LRPAP1 and IFNAR1 were analysed by immunoblotting. Cells were treated with 20 mM MA for 4 h before harvested