Furin is essential for soluble HJV production. (A) HeLa cells were transfected with empty vector (mock), wild type (HJV), and R335Q variant (HJV^R335Q); HJV-transfected cells were treated with 50 μM CMK to inhibit endogenous furin (HJV + CMK). After incubation for 24 hours in serum-free media, media and total lysates (50 μg) were loaded on a 10% SDS-PAGE, blotted, and analyzed by Western blot using the anti-HJV. Anti–α-tubulin was used to verify equal loading. (B) Transfected HeLa cells were fixed and incubated with anti-HJV. The amount of HJV expressed at the cell surface (unpermeabilized cells) is shown as a fraction (percentage) of total protein expression (permeabilized cells) and is indicated as m-HJV (%). Experiments were performed 3 times and made in triplicate. Error bars indicate SD. (C) Transfected HepG2 cells were treated as described in panel A, and media and total lysates were analyzed by Western blot using anti-HJV and anti–α-tubulin. (D) HeLa cells were transiently cotransfected with HJV and furin vectors, both the wild-type (furin) and the inactive variant W547R (furin^W547R). Media and total lysates were analyzed by Western blot using anti-HJV and anti-furin antibodies. s-HJV indicates soluble HJV; c-HJV, cell-associated HJV. The equal loading was verified by α-tubulin. CMK indicates decanoyl-Arg-Val-Lys-Arg-chloromethylketone. Arrows indicate autoproteolytically generated forms; *, unspecific bands. A further aspecific band is recognized by the anti-HJV antibody at approximately 47.5 kDa, as shown in mock lanes. Scales refer to relative molecular mass in kilodaltons.