Figure 5. Determination of the topology of myocilin in the endoplasmic reticulum. The microsomal membrane was prepared from HTM cells transduced with Ad-myocilin-GFP. The membrane fractions were untreated (-) or treated with 1% Triton X-100 (+) followed by the addition of increasing concentrations of protease K (10 μg/ml in lane 1 and 2; 20 μg/ml in lane 3 and 4; 50 μg in lane 5 and 6; 100 μg lane 7 and 8). After digestion, the membrane was probed with anti-myocilin antibody (upper panel), stripped, and re-probed with anti-calnexin antibody (lower panel).