Production of Fluorescent Fusion Proteins

pEGFP–Rab27a was a gift from M. Seabra and described in Hume et al.43 (link) Rab27a was inserted into a pGEMHE–EGFP or pGEMHE–mCherry vector via XhoI and BamHI. pGEMHE–SNAP–Rab11a^S25N was obtained from pGEMHE–EGFP–Rab11a^S25N by switching EGFP with SNAP from pSNAPf (New England Biolabs) via HindIII-XhoI. Full-length pGEMHE–mCherry–myosin-Va, pGEMHE–mCherry–myosin-Va^LT, pGEMHE–mCherry–myosin-Vb^LT, pGEMHE–mCherry–Rab11a, pGEMHE–mCherry–Rab11a^S25N and pCS2-EGFP-UtrCH have been previously described19 (link)20 (link).
These constructs were linearized with AscI (NsiI for Utrophin). Capped mRNA was synthesized using T7 polymerase (mMessage mMachine kit, Ambion), and dissolved in 6-11 μl water. mRNA concentrations were determined using a NanoDrop spectrophotometer system (Thermo Scientific).