Fig. 4

WJ460 directly targets MYOF. a MDA-MB-231 cell lysates were incubated with free biotin or WJ460–biotin. Protein affinity pull-down assay was performed and the precipitated proteins were separated by SDS-PAGE. The gel was processed by silver staining (n = 4). b Exogenous competition assay. MDA-MB-231 cell lysates were incubated with biotinylated-WJ460 (10 μM) or biotin in the presence or absence of 10 (100 μM) or 20 (200 μM) fold WJ460. The biotinylated-WJ460-bound protein was precipitated by streptavidin beads and immunoblotted with an antibody against MYOF (n = 3). c Exogenous competition assay. CHO cells were forced to express human MYOF. Cells overexpressing MYOF were lysed and lysates were incubated with biotinylated-WJ460 (10 μM) or biotin (10 μM) in the absence or presence of 10 (100 μM) or 20 (200 μM) fold WJ460. The mixtures were blotted for MYOF (n = 3). d MDA-MB-231 cells were treated with or without biotinylated-WJ460. Cells were then stained anti-MYOF (labeling MYOF, red) and streptavidin-FITC (labeling WJ460–biotin, green). Scale bars, 10 μm. n = 3. e Transwell invasion assay. Cells were transfected with scrambled siRNA or MYOF siRNA, and then treated with WJ460 (n = 3). Data shown are mean ± s.d. ***p < 0.001, n.s., not significant. f Schematic of constructs with different functional domains of MYOF. g GST-tagged deletion constructs were expressed in BL21 cells. The expressed proteins were incubated with WJ460–biotin or biotin and blotted by anti-GST (n = 3). h Construct containing MYOF C2D was expressed in BL21 cells and the competition assay was performed (n = 3). i Surface plasmon resonance analysis of interactions between WJ460 and MYOF (n = 3). In each panel, n indicates the number of independent experiments performed