Label it nucleic acid labeling kit with cy5

DOTAP, DOPC and DOPE-PEG2000 were purchased from Avanti Polar lipids as chloroform solutions. MVL5 was synthesized as described previously [39 (link)]. The fluorescent lipids TRITC-DHPE and Texas Red-DHPE (Invitrogen, Carlsbad, California) have excitation and emission maxima of 555/589 nm and 580/615 nm, respectively. The RGD-PEG2K-lipid contained a GRGDSP sequence covalently bound to the distal end of PEG2K. It was custom synthesized on solid phase using Fmoc-amino acids and a lipid-PEG2K acid. The pGL3-control vector coding the Luciferase gene (Promega, Fitchburg, Wisconsin) was propagated in E. coli, and purified using a Qiagen Plasmid Mega Prep Kit. The GFP-Rab5-Q79L plasmid was a gift from the Weimbs lab (UCSB) and propagated and purified as decribed above for pGL3. For cell imaging studies, the pGL3 vector was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm) according to the manufacturer’s protocol. For labeling of early endosomes, the CellLights Early Endosome-GFP BacMam 2.0 (Life Technologies, Carlsbad, California) reagent was used according to manufacturer’s protocol.

DOPC was purchased from Avanti Polar Lipids as a solution in chloroform. Pentavalent MVL5,^{88 (link)} PEG2000-lipid,^{26 (link)} RGD-PEG2000-lipid, and RPARPAR-PEG2000-lipid^{76 (link)} were synthesized as previously described. The pGL3 and pGFP plasmids encoding the luciferase and GFP genes were purchased from Promega, and several Rab-GFP plasmids (Rab7,^{89 (link)} Rab9, and Rab11^{90 (link)}) were purchased from addgene.org. Rab5-GFP plasmid was a gift from the Weimbs lab (Molecular, Cellular, and Developmental Biology Department, UCSB). All plasmids were propagated in Escherichia coli and purified using Qiagen Giga or Mega Prep kits. For microscopy studies, the pGFP plasmid was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm). Poly-L-lysine (Sigma-Aldrich) was used to coat glass slides prior to seeding cells for microscopy studies.

DOTAP, DOPC and DOPE-PEG2000 (referred to here as PEG2K-lipid) were purchased as solutions in chloroform from Avanti Polar Lipids (Alabaster, AL). The RGD-PEG2K-lipid contains a GRGDSP peptide (Gly-Arg-Gly-Asp-Ser-Pro-OH) covalently attached to the distal end of the PEG-chain of a custom PEG2000-lipid. It was synthesized via Fmoc solid phase synthesis, employing a lipid-PEG-acid building block in the final coupling step. The chemical structures of the lipids are shown in the Supplementary Material (Fig. S2). TRITC-DHPE (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine) was purchased from Invitrogen and has an excitation and emission maximum of 555 nm and 580 nm, respectively. The luciferase plasmid (pGL3) used in transfection experiments was purchased from Promega. The GFP-tubulin (Clontech) and pGL3 plasmids were propagated in E. coli and purified using a Qiagen Plasmid Mega Prep Kit. For live-cell imaging studies, the pGL3 vector was labeled using the Mirus Bio Label IT Nucleic Acid Labeling Kit with Cy5 (excitation/emission maximum: 649 nm/670 nm) according to the manufacturer’s protocol.