Figure 5.

Expression of Gurken in HeLa cells. A) Plasmids encoding Gurken protein alone or in combination with Myc tagged Star and/or HA tagged Rho were transfected into HeLa cells as indicated on top of the figure or left un-transfected. Aliquots of the cell lysates were applied to SDS-PAGE either directly or after being treated with EndoH. Western bloting was done using an antibody against Gurken. The numbers on the right indicate the molecular mass in kilo Daltons. B-E) HeLa cells were transfected with plasmid generating a double stranded RNA against Sec61β or a control plasmid together with Gurken, Myc-Star and HA-Rho encoding plasmids as indicated. Cells lysates were applied to SDS-PAGE and western blot and probed with an antibody against Sec61β(B). The lysates were also probed with antibodies against Myc and HA to detect expression of Star and Rho respectively (C). The same sets of cells growing on plates were used for a 15 min pulse with ³⁵S methionine and cell lysates were prepared. These lysates were used for western blots with or without EndoH treatment and probed with the Gurken antibody (D). The lysates after the pulse analysis were subjected to immuno-precipitation using anti-Gurken antibody and immuno-precipitated samples were applied to SDS-PAGE either directly or after being treated with EndoH (E). Single glycosylated (0 g) and nonglycosylated (1 g) forms of Gurken are indicated.