Fig. 6. Increased CNIH expression decreases the cell surface level and secretion of TGF. (A) CNIH decreases cell surface TGF in transfected HCA-7 cells, assessed by cell surface protein biotinylation, followed by anti-TGF immunoprecipitation and streptavidin blotting. Duplicate samples were analyzed. Lower panel: total TGF in cell lysates, demonstrating increased relative level of immature TGF (form 1) in the presence of CNIH. (B) CNIH does not affect the cell surface expression of gp130, assessed by cell surface protein biotinylation, followed by avidin precipitation and anti-gp130 blotting. (C) CNIH significantly reduces the level of soluble TGF. Secretion of AP-TGF, measured using an alkaline phosphate assay, in the medium of HeLa cells with or without CNIH. PMA was added to activate shedding. Error bars represent s.e.m.; ^*t-test, P<0.05. (D) CNIH does not affect the subcellular distribution of TACE. Upper panel: cell surface protein biotinylation followed by avidin precipitation and anti-TACE western blot. Lower panel: immunoflorescence staining of HeLa cells. Transfected CNIH-HA is expressed only in transfected cells, whereas endogenous hTACE is expressed in all cells. Bar, 20 µm. (E) Effect of CNIH on N-glycosylation of TGF. HeLa cells were transfected to express TGF with or without CNIH. Cell lysates were treated with N-glycosidase F, and analyzed by SDS-PAGE and western blotting for TGF. Increased mobility after treatment indicates N-glycosylation prior to treatment. CNIH increases the level of immature TGF (form 1) without affecting its N-glycosylation. Dividing vertical white lines indicate parts from the same gel (A).