HJV is secreted in the medium through a BFA-dependent pathway. (A-B) Transfected HeLa cells were incubated for 24 hours in serum-free media. Total lysates (50 μg) and concentrated media were analyzed by using anti-HJV (A) and anti-cMYC (B). (C) R326X- or mock-transfected HeLa cells were incubated in DMEM with 2% FBS; after 2 hours, total lysates and TCA-precipitated media were analyzed by Western blot using anti-HJV. (D) BFA treatment of WT HJV-transfected HeLa cells. HeLa cells were incubated 24 hours with increasing amount of BFA. Concentrated media, PI-PLC supernatant, and total cellular lysates were loaded on 10% SDS-PAGE, and proteins were revealed by anti-HJV. The 50-kDa uncleaved m-HJV was detected by anti-cMYC. The relative molecular mass, in kilodaltons, is indicated on the left.