Cloning and Expression of Endocytic Proteins

ORFs of endocytic proteins were amplified by PCR (Phusion PCR kit; Finnzyme) from IMAGE clones (Geneservice), or directly amplified from cDNA libraries (see Table S2 for details of primers and cDNA sources for the expression constructs used). Each pair of PCR primers was engineered with the appropriate 3′ and 5′ restriction sites for cloning and sequence for either a 9-, 12-, or 13-amino-acid linker between the target protein and FP, as described previously [65] (link). The amplified cDNAs were cloned into mammalian expression vectors in frame with a RFP (in the case of Hip1R, tDimer [65] (link); in the case of myosin1E, mApple [77] (link); and for all other proteins mCherry [78] (link); see Table S2) to generate either N- or C-terminal fusion proteins upon expression.