Figure 2.

Inhibition of GP1 secretion from HEK-293T/17 cells expressing LASV GPC by brefeldin A and monensin. (A) GPC expression in transfected cell extracts. GPC expression was analyzed at 12 (lane 1) and 36 hours (lane 2) post transfection in the absence of inhibitors. Intracellular GPC levels in BFA treated cells at the onset (lane 3), or 12 hours post transfection (lane 4), were compared to monensin treatment at the same time points (lanes 7 and 8, respectively). Vector controls, untreated (lane 5), treated with BFA (lane 6), or monensin (lane 9) only show detection of two endogenous cell proteins (end.). (B) GP1 secretion in corresponding culture supernatants. Significant levels of GP1 were detected only in supernatants of GPC transfected, untreated cells (lane 2), although a minor GP1 band was present in monensin treated cell supernatants at 12 hours post transfection (lane 8). (C) Relative levels of secreted glycoprotein in inhibitor treated conditions were compared to untreated GPC expressed sGP1 at 36 hours (= 1). BFA had a more pronounced effect on GPC and GP1 levels when added at the onset (p < 0.001) (lane 3) than at 12 hours post transfection (lane 4), and resulted in lower levels of expressed glycoprotein than under similar conditions with monensin (lanes 7 [p < 0.05] and 8, respectively). (D) MTT assay, plotted as mean ± SD, N = 3. ANOVA established statistically significant differences (p < 0.01 - 0.001) among several conditions when compared to untreated GPC and vector controls. Inhibitors and time of addition to reactions are outlined below D., and apply to all panels. Lane designations above A. apply to all panels.