Technical Reference #1781

1781.

Rab11-FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport to the endosomal-recycling compartment Conor P. Horgan; Sara R. Hanscom; Rushee S. Jolly; Clare E. Futter; Mary W. McCaffrey, University College Cork, Journal of Cell Science, 123(1781), (2010)
Link To Paper

Keywords:
FIP3; Rab11; dynein; DLIC-1; Rab11-FIP3; DLIC; DYNC1LI1

Materials & Methods:
Plasmid construction: pEGFP-C1/FIP3 pEGFP-C1/FIP3 I738E pEGFP-C3/RCP pTrcHisC/FIP3 pTrcHisC/FIP3 I738E pTrcHisC/FIP2 pTrcHisC/Rip11 and pTrcHisA/RCP have been previously described (Damiani et al. 2004; Horgan et al. 2007; Horgan et al. 2004; Horgan et al. 2005; Kelly et al. 2009; Lindsay and McCaffrey 2004; Lindsay and McCaffrey 2005). To generate pTrcHisC/FIP4 and pEGFP-C1/FIP4 FIP4 was amplified by PCR from an IMAGE clone (7939759) and inserted into the EcoRI site in pGEX-3X (GE Healthcare). The 1.9 kb EcoRI fragment from pGEX-3X/FIP4 was then subcloned into pTrcHisC (Invitrogen) and pEGFP-C1 (BD Biosciences). To generate pGEX-3X/DLIC-1 DLIC-1 was amplified by PCR from an IMAGE clone (40117391) and inserted into the EcoRI site in pGEX-3X. pTrcHisC/DLIC-1 was constructed by subcloning the 1.6 kb EcoRI fragment from pGEX-3X/DLIC-1 into pTrcHisC. pEGFP-C1/FIP3(2-246) was prepared by removal of the 1.6 kb BamHI fragment from pEGFP-C1/FIP3. pEGFP-C1/FIP3(2-435) was prepared by removal of the 1.1 kb KpnI fragment from pEGFP-C1/FIP3. pTrcHisA/FIP3(436-756) was generated by subcloning the 1.1 kb KpnI-EcoRI fragment from pEGFP-C1/FIP3 into pTrcHisA (Invitrogen). pTrcHisB/FIP3(2-435) was generated by subcloning the 1.3 kb BglII-KpnI fragment from pEGFP-C1/FIP3(2-435) into pTrcHisB (Invitrogen). pTrcHisB/ FIP3(247-756) was generated by subcloning the 1.6 kb BamHI-EcoRI fragment from pEGFP-C1/FIP3 into pTrcHisB. pTrcHisB/FIP3(2-246) was generated by subcloning the 0.8 kb BamHI fragment from pTrcHisC/FIP3 into pTrcHisC. pGEX-3X/Rab11a Q70L was generated by subcloning the 0.8 kb BamHI-EcoRI fragment from the previously described pTrcHisC/Rab11a Q70L construct (Horgan et al. 2007) into pGEX-3X. To generate pcDNA3.1HisC/mouse DLIC-1 mouse DLIC-1 was amplified by PCR from pCMV6-mouse DLIC-1 (Origene) and inserted into the XhoI site of pcDNA3.1HisC (Invitrogen). pECFP-C1/DLIC-1 was constructed by subcloning the 1.6 kb EcoRI fragment from pTrcHisC/DLIC-1 into pECFP-C1 (BD Biosciences). pEYFP-C1/FIP3 was constructed subcloning the 2.4 kb EcoRI fragment from pEGFPC1/ FIP3 into pEYFP-C1 (BD Biosciences). To generate pEYFP-C1/RCP RCP was amplified by PCR from an expressed sequence tag template (EST # 3956619) and inserted into the BamHI site of pEYFP-C1. Constructs generated by PCR were confirmed to be correct by double-strand sequencing.

Microscopic Technique
Immunofluorescence and electron microscopy

Cell Type(s)
A431 (epidermal carcinoma) human cell line