Monastrol treatment inhibits PAUF and HRP secretion. (A and B) Secretion of PAUF-MycHis by HeLa cells treated with DMSO (control) or 100 µM monastrol (Mona) was monitored by Western blotting the cell lysate and the medium with anti-His antibody. Single and double star denote the mature and immature form of PAUF, respectively. (B) Quantification of PAUF secretion. The amount of secreted PAUF was normalized to the total cellular levels. The average values of three independent experiments are shown (mean ± SD). Star indicates P < 0.005. (C) HeLa-ssHRP cells were incubated at 37°C with medium containing DMSO or 100 µM monastrol. At the indicated times, the medium was tested for HRP activity by using ECL. Values were corrected for a 21.3% loss of enzymatic activity of HRP by monastrol. (D–F) HeLa-VSV-G-GFP cells were incubated at 20°C for 105 min in the presence of 100 µM cycloheximide, after which the incubation was continued in the presence of DMSO or 100 µM monastrol for 15 min. The cells were then shifted to 32°C for the indicated time. (D) The cells were fixed and incubated with anti–VSV-G antibody without cell permeabilization to specifically detect the cell surface VSV-G-GFP (red) in the total protein (green). Bar, 10 µm. (E) Quantification of the VSV-G trafficking to the cell surface. The percentage of DMSO and monastrol-treated cells with VSV-G-GFP at the plasma membrane (PM) at the indicated time points is shown. The data shown are from a single representative experiment out of two repeats. For the experiment shown, n = 103–124 cells per condition. (F) VSV-G-GFP at the cell surface was monitored by a cell surface biotinylation assay as described in Materials and methods.