Figure 4. Wild-type/mutant myocilin co-expression reduces significantly the extracellular wild-type olfactomedin cleaved fragment of myocilin. HEK-293T cells were transiently transfected with 200 ng of cDNA constructs encoding the 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 (C and E). Ninety-six hours after transfection, recombinant mutant and wild-type myocilins-myc in the culture medium and in cell lysates were analyzed by 10% polyacrylamide SDS-PAGE and western blot using either an anti-myc or a 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 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. The total myocilin in each experiment was calculated as the sum of all the myocilin signals in both the culture medium and the cellular fraction. To facilitate the comparison of the percentage of secreted mutant olfactomedin domain the corresponding bars form panels B and D (G) or panel F (H) are represented together. Error bars represent the S.E. of triplicate experiments. Statistical significance as compared with the COOH-terminal fragment of wild type myocilin was calculated using the Student’s test. The triple asterisk indicates a p<0.001.