Kinesore inhibits granule association of the microtubule motor kinesin-1.
A) Quantitative PCR analysis of mRNA isolated from RBL-2H3 cells. Gene expression level of cargo adaptors known to associate with secretory granules and lysosomes. B) Immunoblot of lysates from RBL-2H3, PC-12, HeLa and HEK293T cells (107 cells/ml lysate). Lysates were probed for Kif5b, and two cargo adaptors, Slp3 and SKIP. SKIP was not detected in RBL-2H3 cells. C) Granule-enriched fractions were prepared by differential centrifugation from unstimulated cells (0’), or from cells that were antigen-stimulated for 15 min and 30 min in the presence of kinesore or vehicle (0.5% DMSO). Fractions were probed by immunoblot for association of the microtubule motor subunit Kif5b, the cargo adaptor Slp3, the granule enzyme rat mast cell protease II (RMCP II) and the mitochondrial marker ATPIF1 (upper panels). Tubulin was not associated with the granule enriched fraction and instead was found in supernatant (lower panels). D) Levels of protein associated with granule-enriched fractions were examined by band densitometry of immunoblots. Values were normalized to unstimulated cells for each experiment (n = 3).
