The furin cleavage of 42-kDa soluble HJV occurs in the ER. (A) Plasma membrane localization of wild-type HJV and HJV^KDEL was quantified using the described binding assay. Experiments were replicated 3 times. Error bars indicate SD. (B) HeLa cells were transiently transfected with mammalian vector encoding the wild-type HJV and HJV^KDEL. Eighteen hours after transfection, sections were stained with anti-cMYC using nanogold protocol as described in “EM and morphometric analysis.” Images were acquired using AnalySIS software (Soft Imaging System, Lakewood, CO). Original magnification, × 23 000. PM indicates plasma membrane; ER, endoplasmic reticulum; G, Golgi; and N, nucleus. (C) Morphometric analysis of wild-type HJV and HJV^KDEL proteins showing the different distribution of the molecules in intracellular compartments. Black bars indicate endoplasmic reticulum (ER); gray bars, Golgi apparatus (G); and white bars, plasma membrane (PM). Error bars indicate SD. (D) Media and total lysates from cells transfected with empty vector (first lane), wild-type HJV (second lane), HJV^KDEL (third lane), and HJV^KDEL plus 50 μM CMK (fourth lane) were analyzed by Western blot and anti-HJV. Anti–α-tubulin was used to verify equal loading. s-HJV indicates soluble HJV; c-HJV, cell-associated HJV; and CMK, decanoyl-Arg-Val-Lys-Arg-chloromethylketone. (E) HeLa cells were transfected with the empty vector (mock), HJV^KDEL, and the autoproteolytically defective variant W191C ^KDEL; total lysates (50 μg) were loaded on a 10% SDS-PAGE, blotted, and analyzed using anti-HJV. The arrow indicates the autoproteolytically generated fragment. The relative molecular mass, in kilodaltons, is indicated on the left.