Fig 5. Inhibition of AR prevents LPS-induced decrease in the expression of Na/K-ATPase transporter protein in hNPECs.

Growth-arrested hNPECs were preincubated with zopolrestat for 24 h followed by incubation with LPS (1 μg/ml) for additional 24 h. At the end of incubation, cells were harvested and either lysed directly (A) or membrane fraction was prepared (B) and equal amount of protein was separated on SDS-PAGE followed by immunoblotting using antibodies against alpha subunit of Na/K-ATPase. Same blots were stripped and immunoblotting using antibodies against GAPDH (A) and cadherin (B) was performed to show equal loading of protein. Numbers below the blots show fold change in the expression of proteins (n=3). (C) For immuno-cytochemistry hNPECs, after incubation with LPS for 24 h, were fixed in prechilled methanol (at −20°C) and air-dried. The cells were washed with cold PBS and immuno-cytochemistry using antibodies against Na/K-ATPase protein was performed using standard protocol. A representative (n=3) fluorescent photomicrograph and corresponding DAPI images have been shown from each group. (D) Pixel density was measured from fluorescent photomicrograph in (C) and corresponding values are shown as bar diagram. ^*p<0.01 Vs Control; ^**p<0.01 Vs LPS alone (n=3). (E) Serial sections of paraformaldehyde-fixed rat eyes were immuno-stained with antibodies against alpha subunits of Na/K-ATPase protein. The expression of Na/K-ATPase was observed in the pars plicata region of ciliary bodies of LPS-treated rat. A representative (n=4) photomicrograph is shown from each group. sorb, sorbinil; zopol, zopolrestat.