Figure 1. Electrophoretic mobility and glycosylated status of intracellular and extracellular wild-type myocilins. HTM cells were plated into a 24 well plate, grown to 70% confluence, and transduced for 2 h with Ad-myocilin-FLAG at an MOI of 10 pfu. After 48 h, the cells and culture media were harvested and the myocilin in the samples were analyzed by western blot using polyclonal anti-myocilin antibody. A: Cells and culture media were harvested together (lane 1), or cells and culture media were separately harvested (lane 2 and 3), or cells were replenished after culture with fresh culture media and then cells and culture media were harvested together (lane 4), or cells were cultured with serum-free culture media and then the conditioned media was harvested (lane 5). Cells were harvested with 0.4 ml of 1X Laemmli sample buffer whereas culture media (0.3 ml) was harvested with 0.1 ml of 4X Laemmli sample buffer. B: Myocilin in cells (lane 2 and 3) or media (lane 5 and 6) was treated with Endo H (lane 2 and 5) or PNGase F glycosidase (lane 3 and 6) as described in Methods. Undigested myocilins in cells (lane 1) and culture media (lane 4) served as controls. C: Myocilin in cells was either treated (lane 1) or not treated (lane 2) with Endo D glycosidase.