Figure 5. Aggregation of wild-type and mutant myocilins co-expressed for 24 h. HEK-293T cells were transiently transfected with cDNA constructs encoding 5 different myocilin mutants or wild-type myocilin as described in Figure 2. Myocilin aggregates secreted after 24 h of expression were analyzed by nonreducing SDS-PAGE (4-15% gradient polyacrylamide) and were detected by western blot. The culture media from co-transfections were analyzed using either an anti-myc antibody to detect mutant myocilin (A), or an anti-HA antibody to detect wild-type myocilin (B). Control single transfections were analyzed using an anti-myc monoclonal antibody (C). Molecular weight markers are shown on the right of panels A, B and C. To facilitate the comparison of the molecular size of myocilin aggregates different volumes (1 to 50 μl) of culture medium from panels B (D) and C (E) were loaded into gels and analyzed by nonreducing SDS-PAGE (8% polyacrylamide).