Figure 1: Rab10 interacts with MYO5B via exon D.

(a) Lysates of cultured cortical neurons were subject to IP with MYO5B antibody or control IgG, followed by IB with antibodies against Rab10 or MYO5B. (b) Transcription of MYO5A and MYO5B isoforms in the P0 rat brain. The amplified 411 bp and 333 bp bands in RT-PCR represent MYO5B isoforms with (+) and without (−) exon D, respectively. (c) Exon D of MYO5B is conserved among different species indicated by the amino acids alignment. (d) Cell lysates of HEK 293 cells transfected with HA-Rab10 and GFP-MYO5B without or with exon D were subject to IP with HA antibody, and then IB with HA or GFP antibody. (e) GST-Rab10 immobilized on glutathione Sepharose beads was preloaded with GTPγS or GDP, and then incubated with purified His6-exon D. Bound proteins were subject to IB with His antibody. (f) His6-ExonD immobilized on Ni-NTA agarose was incubated with lysates of cultured cortical neurons. Beads-associated proteins were determined by IB with indicated antibodies. (g) Cultured hippocampal neurons were co-transfected with TD-Rab10 and GFP-MYO5B (+D) or MYO5B (−D). At DIV3, transfected neurons were imaged to determine the localization of Rab10 and MYO5B isoforms. The enlarged areas show perinuclear regions with a large fraction of Rab10 colocalized with MYO5B (+D), compared to a lesser extent with MYO5B (−D). (h) Quantification for the proportion of Rab10 vesicles colocalized with MYO5B vesicles (see detailed information in Methods). Data are shown as mean±s.e.m. At least 20 neurons from each group were analyzed. ***P<0.001, Student’s t-test. Scale bar=20 μm.