Plasmid Construction and Characterization

The plasmid pOTB-UBIAD1 was purchased from Open Biosystems Inc. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described^{28 (link)}. For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector. BFP-H-Ras was constructed by fusing H-Ras at the N-terminus of BFP into the pcDNA3.1 vector. GFP-H-Ras and its mutation were cloned into pEGFP-C1. Flag-H-Ras and its mutation were constructed by fusing the Flag tag to the N-terminus of H-Ras and inserting into pcDNA3.1. The plasmid 3Flag-UBIAD1 was constructed by fusing the 3Flag tag to the N-terminus of UBIAD1 and inserting into pcDNA3.1. pGFP-RBD was kindly provided by Dr. Mark R. Philips. Full-length human UBIAD1 and Apo E cDNA fragment were separately cloned into the pEGFP-N1 vector to create UBIAD1-EGFP and Apo E-EGFP. UBIAD1 was cloned into pcDNA3.1 to generate pcDNA3.1-UBIAD1. UBIAD1-BFP, Golgi-BFP and Apo E-BFP were constructed by fusing BFP with the C-terminus of UBIAD1 and Apo E and inserting into pcDNA3.1. All vectors for the BIFC assay were constructed using pcDNA3.1.