Fig. 4

HTT, HIP1, Rab11-FIP5 and motors associate with isolated Rab4-vesicle membranes. a Schematic diagram of mouse brain homogenate fractionation into perinuclear supernatant (PNS), vesicle fraction (VF), soluble, and heavy membrane (P1) fractions by ultra-centrifugation and sucrose gradient separation. b Representative western blot of each fraction probed with antibodies to synaptotagmin-1 (SYT1, VF), synaptophysin (SYP), KDEL (ER), Golgi, Cytochrome C (mitochondria), Actin and Tubulin (loading controls), Rab4, HTT, KIF5C (kinesin), DIC (dynein), HIP1, Rab11-FIP5, HAP1, Rab11, Rab5 or Syntaxin17 (SYX17). Rab4, SYT1, SYP, SYX17, Rab5, and Rab11 are enriched in the VF, while HTT, HIP1, HAP1, Rab11-FIP5, KIF5C and DIC are present in the VF and the soluble fraction. KDEL, Golgi and cytochrome C are enriched in P1. c Representative western blot of an immunoprecipitation of Rab4-containing VF, probed with HTT, Rab5, Rab11, and HAP1. HTT and Rab11 show their presence in the Rab4 IP, but HAP1 is not seen. No bands are seen in the negative no antibody control (−Ctrl). d Representative western blot of an immunoprecipitation of Rab4-containing VF, probed with DIC, HIP1, Rab11-FIP5, SYP, SYT1, and SYX17. DIC, HIP1, Rab11-FIP5, SYP, and SYT1 are all present in the Rab4 IP, but not SYX17. No bands are seen in the negative no antibody control (−Ctrl). e Representative western blot of an immunoprecipitation of Rab4-containing VF, probed with KIF5C. KIF5C show that these are present in the Rab4 IP. n = 3