Research Paper Volume 16, Issue 20 pp 13039—13075

Figure 1. JESS capillary-based Westerns demonstrate canonical EphA2-pY589 activation is restricted to the lens epithelial cells while non-canonical EphA2-pS898 phosphorylation is present in epithelial cells and cortical fibers of 6-week-old, 4-month-old, and 8-month-old ephrin-A5^+/+ and ephrin-A5^-/- mice. Representative gel bands for pan-EphA2 (~128kDa), EphA2-pY589 (~128kDa), EphA2-pS898 (~128kDa), and total protein profiles (12-230kDa) in lens epithelial (A) and cortical fiber cell fractions (B) are shown. The dot plots display the average and standard deviation of amounts for proteins of interest normalized to the amount of total protein in each sample. Black horizontal lines mark changes between ephrin-A5^+/+ samples at different ages, and blue horizontal lines mark changes between ephrin-A5^-/- samples at different ages. Red horizontal line marks changes between ephrin-A5^+/+ and ephrin-A5^-/- samples. *, P< 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. N = 3 biological replicates per group. (A) In epithelial cells, the amount of pan-EphA2 protein decreases with age, and the levels are comparable between ephrin-A5^+/+ and ephrin-A5^-/- samples. Canonically active EphA2-pY589 protein is detected in all samples, and there is no change with age or genotype. Unexpectedly, non-canonically active EphA2-pS898 protein is detected in both ephrin-A5^+/+ and ephrin-A5^-/- samples. In ephrin-A5^+/+ epithelial cells, there is increase in EphA2-pS898 levels between 6-week-old and 4-month-old or 8-month-old samples, and there is no change between 4-month-old and 8-month-old control samples. In ephrin-A5^-/- epithelial cells, there is continuous increase of EphA2-pS898 levels with age, and there is a significant increase in EphA2-pS898 level in 8-month-old ephrin-A5^-/- samples compared to that in control samples. (B) In cortical fiber cells, pan-EphA2 levels decrease between 6-week-old and 4-month-old or 8-month-old samples, and there is no change between 4-month-old and 8-month-old control samples. There are no differences in pan-EphA2 levels between ephrin-A5^+/+ and ephrin-A5^-/- samples. Canonically active EphA2-pY589 protein was not detected in cortical fiber cells. Non-canonically active EphA2-pS898 protein levels increase with age in ephrin-A5^+/+ and ephrin-A5^-/- samples. The levels of EphA2-pS898 proteins are not changed by the loss of ephrin-A5. The amount of pan-EphA2 decreased after 6-weeks but EphA2-pS898 amounts increased with age.