Figure 4. Western immunoblot of two novel myocilin mutations found in this study and expressed in transiently transfected 293T cells

Two hundred nanograms of DNA constructs encoding myc epitope-tagged versions of mutant myocilin forms (Arg346Thr, Tyr479His, Gln368Stop and Pro370Leu) were transfected into 293T cells. Separation of culture medium, soluble cellular fractions, and insoluble cellular fractions were carried out as indicated in the Materials and Methods. Detection was performed with an anti-myc monoclonal antibody. Myc-tagged wild-type myocilin was used as a control of normal expression and the myocilin mutation Pro370Leu was employed as a control of disease-causing mutation. The arrow and arrowhead indicate the position of the 55 kDa and 35 kDa myocilin bands, respectively. c.m.: culture medium; s.c.f.: soluble cellular fraction; i.c.f.: insoluble cellular fraction.