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. 2023 Jul 25;12:e85878. doi: 10.7554/eLife.85878

Figure 1. YBX1 directly and specifically binds miR223.

(a) RT-qPCR analysis of fold change of miR-223 and miR-190 in cells and purified exosomes from 293 T WT cells and YBX1 knockout cells. Data are plotted from three independent experiments, each independent experiment with triplicate qPCR reactions; error bars represent standard deviations. (b–c) EMSA assays using 1 nM 5’ fluorescently labeled miR223 or miR190 and purified YBX1. Purified YBX1 was titrated from 500pM to 1 μM. In gel fluorescence was detected. Quantification of (d) shows the calculated Kd. (e) Schematic diagrams of the different domains of YBX1. (f) EMSA assay using 1 nM 5’ fluorescently labeled miR223 and purified YBX1 truncations. [YBX1(1–51) or YBX1(52–129) or YBX1(130–324).] (g) EMSA assay using 1 nM 5’ fluorescently labeled miR223 and purified YBX1 truncations [YBX1(1–129) or YBX1(52–324)] or YBX1(F85A) mutant.

Figure 1—source data 1. Uncropped gel images corresponding to Figure 1.

Figure 1.

Figure 1—figure supplement 1. Knockout of YBX1 did not change exosome secretion.

Figure 1—figure supplement 1.

(a) Exosome numbers are measured by Nanosight NS300. Normalized exosome number from 293 T WT and YBX1-KO cells are shown.
Figure 1—figure supplement 2. Purified YBX1 full length protein and different truncations and mutation.

Figure 1—figure supplement 2.

(a–c) YBX1 full-length protein or different truncations were overexpressed and purified from insect cells.
Figure 1—figure supplement 2—source data 1. Uncropped gel images corresponding to Figure 1—figure supplement 2.