Eg5 T112N expression inhibits PAUF secretion and causes accumulation of CARTS. (A and B) HEK 293T cells were transfected with a plasmid for PAUF-MycHis (control) or plasmids for Myc-Eg5 T112N and PAUF-MycHis. 5 h after transfection, 2 mM thymidine was added to the medium, and 18 h later the cells were washed and incubated with fresh medium. At the indicated time points, the medium was Western blotted with anti-His antibody. (B) Quantification of PAUF secretion. The amount of secreted PAUF was normalized with the total cellular levels. The data shown are from a single representative experiment out of three repeats. (C and D) HeLa cells were cotransfected with plasmids for Myc-Eg5 T112N and PAUF-mRFP and visualized by fluorescence microscopy. The cells expressing PAUF-mRFP alone were observed as a control. Bars, 10 µm (inset, 5 µm). (D) Quantification of PAUF containing CARTS. The number of CARTS in control cells (n = 1,946 punctate elements in 20 cells) and Myc-Eg5 T112N–expressing cells (n = 3,701 punctate elements in 20 cells) was determined. The average number of CARTS per 100 µm² is shown (mean ± SD). Star indicates P < 0.001. (E and F) HeLa cells were cotransfected with plasmids for Myc-Eg5 T112N and VSV-G-GFP. The cells were incubated at 20°C for 2 h in the presence of 20 µg/ml cycloheximide, shifted to 32°C, and fixed at the indicated time points. The cells expressing VSV-G-GFP alone were observed as a control. Bar, 10 µm. (F) Quantification of the VSV-G trafficking to the cell surface. The percentage of control and Myc-Eg5 T112N–expressing 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 = 55–58 cells per condition.