Figure 2.

Glycosylation differences of select target proteins in glaucomatous TM compared to controls (n= 12 each) and in response to glycosylation inhibitors. (A) Representative Western analyses of Keratocan in control and glaucomatous TM protein extracts (10μg) with or without PNGase F treatment as indicated. The ratio of intact to collapsed protein band on digestion provided the ratio of glycosylation for normal or glaucoma. The ratio of intact band between glaucoma to normal provided the level of protein (normalized to total protein, GAPDH or Beta-actin). (B) Representative Western analyses of Biglycan in control and glaucomatous TM protein extracts with PNGase F treatment. (C) Representative Western analyses of Biglycan in control and inhibitor treated (SS: 150μM, PhQ: 75 μM, MeQ: 50 μM) cell extracts subjected to PNGase F treatment. Glycerol 3-phosphate dehydrogenase (GAPDH) immunoreactivity as indicator of loading control for panels A through C have been presented as indicated. (D) Densitometric estimation of proteins (hollow bars) and glycosylation (hashed bars) expressed as ratio for each specific protein as indicated. The ratios for proteins and glycosylation are immunoreactivity in glaucoma/normal, except for Biglycan†, which indicates ratio of inhibitor treated/untreated control. The glycosylation level in normal or glaucoma was the normalized ratio of glycosylation estimated by partial digestion of PNGase treatment compared to undigested control band (as shown for Keratocan in A). The overall ratio of glycosylation estimated in glaucoma to control (for Biglycan†, inhibitor treated to control) using this procedure has been depicted with hashed bars. The results are mean± SD of three independent experiments. The mean ratio was subjected to a two-tailed paired t-test; *P ≤0.05.