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. 2016 Jul 29;7(42):68057–68071. doi: 10.18632/oncotarget.10929

Figure 3. NRP1 colocalization with miRNA conjugated to the beads, unassisted NRP1 knockdown, and binding control.

Figure 3

A. Colocalization index. The experiment was performed as in Figure 2. In the cytoplasm 90% of miR-422 is colocalized with NRP1 as estimated by the calculation of Manders' coefficient (M1), and this was blocked by anti-NRP1 antibody. The data represents the Mean±SEM; **** P < 0.0001. B. NRP1-positive BT-474 breast cancer cells treated with NRP1-targeting siRNA without transfection reagent express significantly less NRP1 than the cells treated with scrambled siRNA. After 3 days in culture the cells were fixed, permeabilized, stained for NRP1, and analyzed by flow cytometry. The data is representative of three independent experiments. C. The streptavidin-coated microbeads did not bind significantly to NRP1-Fc, but binding was enhanced by pre-incubating the NRP1-Fc coated plate with a biotin-conjugated miRNA. Retention of the beads was quantified by fluorescent ELISA. Three experiments yielded similar results; the data represents Mean±SEM, *. P < 0.05.