JIP3/JIP4 regulate KIF5B and p150Glued but not KIF3A association to MT1-MMP endosomes. (A–D) Association of indicated motor proteins (green and red) with MT1-MMPmCh–positive endosomes. Arrows in B point to JIP4 and p150Glued colocalization. (E) MDA-MB-231 cells expressing MT1-MMPpHluorin analyzed by in situ PLA (red) with p150Glued and JIP4 antibodies. Bars, 2 µm. (F) PLA signal in siRNA-treated cells using the indicated antibodies (in brackets). Values are mean number of PLA dots/cell ± SEM. n, number of cells analyzed for each cell population. (G) MDA-MB-231 cells expressing MT1-MMPmCh treated with the indicated siRNAs and stained for KIF5B and p150Glued (top) or KIF5B and KIF3A (bottom). Bar, 2 µm. (H) Motor protein association (KIF5B, p150Glued, or KIF3A) with MT1-MMP–positive compartments compared with association in siNT-treated control cells (see Materials and methods section 3D deconvolution microscopy). n, number of endosomes analyzed for each cell population. (I) Detection of KIF5B and KIF3A endogenous proteins in anti–MT1-MMP immunoprecipitates (IPs) from MDA-MB-231 cells. (J) Detection of MT1-MMPmCh in anti-GFP IPs of YFP, KIF5B-YFP, or KIF3A-GFP. (K) Detection of KIF5B, p150Glued, and KIF3A motor proteins in MT1-MMPmCh IPs from MDA-MB-231 cells transfected with the indicated siRNAs. (L) Quantification of motor association from immunoblotting analysis as in I. Levels in siNT control cells were set to 100. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (as compared with siNT-treated cells).
