Identification of the myocilin region involved in its intracellular accumulation in response to extracellular bicarbonate-depletion.
2013-01-16T02:18:49Z (GMT) by
<p>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 <i>A, C, E, and G,</i> 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.</p>