The GFP-KDEL was constructed by adding the nucleotides encoding the amino acids KDEL (5′-AAAGATGAGTTG-3′) to the C-terminal end of the GFP open reading frame. The clathrin light chain was cloned into the EcoRI and SalI sites of the pEYFP-C1. The GFP-Lact-C2 and mRFP-Lact-C2 were constructed first by PCR amplifying the bovine C2 domain of Lactadherin and subsequently cloned into the pEGFP-C1 and mRFP-Lact-C2 vectors, respectively, using the BglII and EcoRI sites (Touret et al., 2005 (link); Yeung et al., 2008 (link)). CD63-mCherry was a gift from W. Trimble (Hospital for Sick Children, Toronto, Ontario, Canada). GalT-GFP was provided by E. Rodriguez Boulan (Weill Cornell Medical College, New York, NY). The 2xFYVE-RFP was provided by L. Cantley (Harvard University, Cambridge, MA), and the GFP-p40-PX by M.B. Yaffe (Massachusetts Institute of Technology, Cambridge, MA). The PH domain of PLCδ was provided by T. Balla (National Institutes of Health, Bethesda, MD) and the VSVG-GFP ts045 construct was provided by J. Lippincott-Schwartz (National Institutes of Health). Sec61α-GFP (Greenfield and High, 1999 (link)), RFP-Rab5 (Vonderheit and Helenius, 2005 (link)), and Lamp1-RFP (Sherer et al., 2003 (link)) were described earlier and mito-RFP was obtained from Invitrogen. The TGN marker GCC88-myc was provided by J. Stow (Institute for Molecular Bioscience, the University of Queensland, Brisbane 4072 QLD, Australia) and described previously (Luke et al., 2003 (link)).