Analysis of the autoproteolytic cleavage and the cell-surface expression of HJV. (A) HeLa cells were transfected with empty vector (mock)–, WT-, and mutant HJV–expressing constructs; 50 μg of total lysates were loaded onto a 15% SDS-PAGE, blotted, and incubated with anti-HJV. Full-length (approximately 50 kDa) and cleaved (approximately 33 kDa) bands are indicated. *Unspecific bands. (B) Transfected HeLa cells were fixed and incubated with anti-cMYC. The amount of HJV expressed at the cell surface (nonpermeabilized cells) is shown as a fraction (percentage) of total protein expression (permeabilized cells). Statistical significance was calculated on 6 independent experiments, made in triplicate. *P < .001. (C) Transfected HepG2 cells were treated as described in panel A, and analyzed for the percentage of cell-surface HJV. *P < .001. (D) Top panel: transfected HeLa cells were incubated with (+) or without (−) PI-PLC (0.3 U/mL) in PBS at 37°C; after 2 hours, TCA-precipitated proteins were analyzed with anti-HJV. Bottom panel: transfected cells were incubated for 2 hours in DMEM containing 0.3 U/mL PI-PLC. Total lysates and acetone-precipitated proteins were analyzed under mildly reducing conditions with anti-HJV. Scales refer to relative molecular mass in kilodaltons. Error bars indicate SD.