Identification of the myocilin region involved in its intracellular accumulation in response to extracellular bicarbonate-depletion.

HEK-293T cells (400000 cells/plate in 24-well plates) were transfected with cDNA constructs encoding full-length myocilin (A), a myocilin N-terminal fragment (amino acids 1–226) (C), a myocilin C-terminal fragment (amino acids 217–504) (E) or the control protein PEDF (G). All the recombinant proteins were fused to the myc epitope on their C-terminal ends. After transfection, cells were incubated for 48 h in 3 different culture medium volumes (500, 350 and 180 ul). Extracellular and intracellular recombinant proteins were analyzed in aliquots representing 5% of either the total culture medium volume or cell lysate, by 10% polyacrylamide SDS-PAGE and Western blot using an anti-myc monoclonal antibody. As an internal control of sample loading or cell integrity LDH was detected in cell extracts and culture media, respectively, using a goat anti-LDH antibody. (B, D, F and H). Quantitation by densitometry of the recombinant proteins detected in A, C, E, and G, respectively. Error bars correspond to the SD of three independent experiments carried out in triplicate. One-way ANOVA analysis for extracellular full-length myocilin, extracellular C-terminal fragment and intracellular myocilin in B, p<0.01, p<0.01 and p = 0.05, respectively. One-way ANOVA analysis for extracellular and intracellular N-terminal fragment in panel D, p<0.01 and p<0.001, respectively. One-way ANOVA analysis in F and H, p>0.05.