Figure 2. Wild-type/mutant myocilin co-expression significantly increases the amount of extracellular mutant myocilin compared to single mutant expression. HEK-293T cells were transiently transfected with 200 ng of cDNA constructs encoding 5 different myocilin-myc mutants or wild-type myocilin-myc (A). To model the heterozygous expression of myocilin mutants, cells were co-transfected with 200 ng of cDNAs encoding each mutant myocilin-myc and 200 ng of a cDNA construct encoding wild-type myocilin-HA. Twenty-four hours after transfection, the recombinant mutant-myc (C) and wild-type-HA myocilins (E) in both the culture medium and cells were analyzed by 10% polyacrylamide SDS-PAGE and western blot using either an anti-myc or an anti-HA monoclonal antibody, respectively. Arrows and arrowheads indicate the position of full-length myocilin (55 kDa), and the 35 kDa processed olfactomedin-containing fragment, respectively. Molecular weight markers are shown on the left. B, D and F: Densitometric quantitation of full-length myocilin and its processed olfactomedin-containing fragment, detected in A (bars with oblique lines), C (solid bars), and E (dotted bars). Red bars represent the percentage of extracellular unprocessed full-length or truncated Q368X myocilin (extracellular myocilin/total myocilin), blue bars correspond to the extracellular olfactomedin-containing fragment (extracellular 35 kDa myocilin/total myocilin), and yellow bars represent intracellular full-length myocilin or the truncated Q368X form (intracellular myocilin/total myocilin), respectively. Total myocilin in each experiment was calculated as the sum of all the myocilin signals in both the culture medium and the cellular fraction. Error bars represent the S.E. of triplicate experiments. To facilitate the comparison of the percentage of extracellular unprocessed full-length or truncated Q368X myocilin, the corresponding bars form panels B and D (G) or panel F (H) are represented together. Numbers above bars in panel G indicate the fold-increase of full-length myocilin in cotransfections versus single transfections. Statistical significance as compared with full-length wild-type myocilin, was calculated using the Student’s test. The asterisk indicates a p<0.05, the double asterisk indicates a p<0.01 and the triple asterisk indicates a p<0.001.