Figure 7.

ER-Golgi transport of VSVG-tsO45-YFP is delayed in p125A-silenced cells. (A) HeLa cells were transfected with VSVG-tsO45-YFP and incubated at nonpermissive temperature at 40°C for 24 h. 5 min after release from nonpermissive temperature, the cells were fixed and double labeled with rabbit anti-p125A and mouse anti-Sec31A antibodies. The bottom panels represent the enlarged view of the boxed area in the top panels. The merged images are shown in d and h. Arrowheads indicate colocalization of p125A, Sec31A, and VSVG-tsO45-YFP. Bar, 10 µm. (B) HeLa cells were transfected twice with ON-Targetplus SMARTpool siRNA (Thermo Fisher Scientific) against p125A or nontarget control. 48 h after the initial transfection, the cells were transfected again to express VSVG-tsO45-YFP at 40°C overnight. At 72 h, the cells were released from 40°C block and incubated at 32°C for various times as indicated. Cells were then processed to view VSVG-tsO45-YFP. Bar, 10 µm. (C) VSVG-tsO45-YFP acquisition of Endo H resistance is delayed in p125A knockdown cells. HeLa cells silenced of p125A were transfected with VSVG tsO45-YFP plasmid and incubated at 40°C overnight. To allow VSVG-tsO45-YFP transport, the cells were shifted to 32°C. At the indicated times, cell lysate was prepared and subjected to Endo H treatment followed by SDS-PAGE and immunoblotting. R and S denote Endo H–resistant and Endo H–sensitive forms, respectively. The ratio (percentage) of the amount of Endo H–resistant form to that of the total amount (Endo H–resistant + Endo H–sensitive form) was plotted below. Green solid line represents kinetics of acquisition of VSVG tsO45-YFP in nontarget siRNA-treated cells and red dashed lines indicated that in p125A-silenced cells. (D) Secretion of secreted alkaline phosphatase (SEAP) is retarded in p125A-silenced cells. HEK293 cells stably expressing SEAP were transfected with ON-Targetplus SMARTpool siRNA (Thermo Fisher Scientific) for p125A or nontarget control as described in Materials and methods. 72 h after the initial transfection, the cells were washed five times with fresh media (prewarmed to 37°C) and allowed to secrete for various times. Volumes equivalent to 10% of the media were collected at 0, 15, 30, 45, 60, 90, and 120 min. After 120 min, the cells were washed twice with PBS and lysed with 1X SDS sample buffer. 2% cell lysate were analyzed by SDS-PAGE, and then subjected to immunoblot analysis using antibodies against p125A and actin. The collected media was subjected to Chemiluminescent SEAP Assay as described by manufacturer (Takara Bio Inc.) to quantify the amount of SEAP present. The results were then normalized by actin and plotted against time. The experiment was repeated three times. P < 0.0001. Molecular size markers are in kD.