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. 2018 Feb 15;10(3):1075–1087. doi: 10.1016/j.stemcr.2018.01.017

Figure 2.

Figure 2

High- and Low-Phagocytic RPE Lines Do Not Differ in Expression of Phagocytic Receptor Proteins αv Integrin, β5 Integrin, or MerTK or in Apical Secretion of Phagocytic Ligands MFG-E8 and Protein S

(A) Representative immunoblot (showing samples from lines #1 and #5) probed sequentially for proteins as indicated.

(B) Quantification of immunoblotting experiments performed as in (A) for all eight RPE lines. Dot plots show relative protein expression as indicated by high-phagocytic lines #1–4 (circles) and low-phagocytic lines #5–8 (triangles). Each protein was quantified compared with tubulin as a loading control and relative to a standard included on all gels, and consisting of a mixed-cell line sample; symbols indicate mean of n = 3 independent samples for each line. Student's t test did not show significant differences between the averages of high- and low-phagocytic RPE cells.

(C) Absolute levels of secreted MFG-E8 in ng/mL measured by ELISA in supernatants collected from the apical (ap) and basal (bl) chambers of transwell inserts of RPE cells as indicated. Supernatants were collected 72 hr after complete medium change. Error bars show mean ± SEM, n = 4 high-phagocytic lines (black bars, ∗p < 0.05) and 4 low-phagocytic lines (gray bars, ∗p < 0.01). Asterisks indicate significant difference of apical and basal MFG-E8 content as indicated by Student's t test.

(D) Representative protein S immunoblot supernatants retrieved from apical and basal chambers of high-phagocytic RPE line #1 and low-phagocytic RPE line #5, as indicated, after 4 weeks of culture. Non-specific protein stain of the membrane before block is shown to demonstrate similar load. Left bar graph shows relative total (apical plus basal) secreted protein S levels of the 4 high-phagocytic lines (black bars) and the 4 low-phagocytic lines (gray bars) quantified compared with a standard of purified human protein S (Hyphen Biomed) included on all gels, mean ± SEM. Right bar graph shows percentage of apical protein S of total protein S, mean ± SEM, n = 4 high-phagocytic lines (black bars) and 4 low-phagocytic lines (gray bars). Differences were not significant.