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Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

Fig 5

Topical CLU binds selectively to the ocular surface subjected to desiccating stress, and to LGALS3 in vitro.

(A) The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. (B) A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. (C) LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose

Fig 5