Since several antibodies for ER stress markers did not work on paraffin-embedded TM sections, we next examined ER stress markers by Western blot analysis of human TM tissue lysates. Western blot analysis demonstrated that GRP78, GRP94, ERO-1α, ATF-4, and CHOP were increased in the age-matched glaucomatous TM (POAG) tissues compared to normal TM lysates (
Figs. 3A–G). Both phosphorylated EIF-2α (
Figs. 3A,
3G) and total EIF-2α (data not shown) levels were not changed in the glaucomatous TM compared to normal TM tissues. Densitometric analysis of Western blots for ER stress markers normalized to loading control β-actin is shown in
Figures 3B through 3G. GRP78 and GRP94 levels were increased in the glaucomatous TM lysates, but the differences were not statistically significant (for GRP78, 0.0022 ± 0.0006 in normal versus 0.016 ± 0.008 in glaucoma,
P = 0.087; for GRP94, 0.04 ± 0.006 in normal versus 0.2 ± 0.1 in glaucoma
P = 0.1007;
n = 4 normal and 5 glaucoma;
Figs. 2B,
2C). However, markers of chronic ER stress including ERO-1α, ATF-4, and CHOP were significantly increased in the glaucomatous TM lysates (for ERO-1α, 0.0006 ± 0.0002 in normal versus 0.002 ± 0.0008 in glaucoma,
P = 0.0247; for ATF-4, 0.002 ± 0.001 in normal versus 0.019 ± 0.005 in glaucoma,
P = 0.0129; and for CHOP, 0.005 ± 0.002 in normal versus 0.05 ± 0.02 in glaucoma,
P = 0.0294;
n = 4 normal and
n = 5 glaucoma;
Figs. 2D–F). No change was observed in phosphorylated pEIF-2α levels (0.005 ± 0.001 in normal versus 0.007 ± 0.003 in glaucoma,
P = 0.289). Together, immunostaining and Western blot data indicate the presence of chronic ER stress in the glaucomatous TM.