Fluorescent Labeling of Endocytic Proteins

The plasmids encoding Rab5a, Rab7a, and LAMP1 wt and mutant molecules tagged with EGFP have all been described previously [19 (link)]. Stock solutions of chloroquine diphosphate (Sigma) and ammonium chloride (NH₄Cl, Sigma) were prepared in dH₂O at concentrations of 19 mM and 1 M, respectively. Bafilomycin A1 (BioViotica), colcemid (Cayman Chemical), concanamycin B (BioViotica), MG-132 (Selleck Chemicals), nocodazole (Merck), and taxol (Merck) were all dissolved in 100% dimethyl sulfoxide (DMSO) to prepare stock solutions at 100 μM, 10 mM, 50 μM, 40 mM, 20 mM, and 10 mM, respectively. Stock solutions of all drugs were diluted in DMEM at the indicated doses (Figs 4 and 5), which are known not to cause cytotoxicity [23 (link), 34 (link)]. Both dH₂O and DMSO were included as solvent controls. The hydroxysuccinimidyl (NHS) ester dyes AF488 (Thermo Fisher Scientific) and ATTO647N (Atto-Tec) were dissolved in DMSO (10 and 5 mg.mL^-1, respectively) while octadecyl rhodamine B chloride (R18, Thermo Fisher Scientific) was dissolved in ethanol (10 mM).