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. 2020 Jan 21;219(3):e201908142. doi: 10.1083/jcb.201908142

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© 2020 He et al.

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Figure S6. — In vitro disassembly of clathrin/AP2 coats and sCCVs containing PtdIns(3)P or PtdIns(4)P. (A) SDS-PAGE (and Coomassie blue staining) of the recombinant full-length Aux1, ΔPTEN-Aux1, and full-length GAK. Molecular weight markers are shown. For GAK and ΔPTEN-Aux1, impurities (of high electrophoretic mobility relative to the target species) reduced the full-length target protein proportion to 60 and 50%, respectively (estimated by band densitometry). (B) Single-object uncoating efficiency determined from the loss of the clathrin LCa–Alexa Fluor 488 fluorescence signal as a function of ΔPTEN-Aux1, full-length Aux1, or full-length GAK concentration (5–25-nM range) added together with 1 µM Hsc70 and 5 mM ATP. Each sample included a mixture of clathrin/AP2 coats (CC) together with sCCVs containing PtdIns(4,5)P₂ together with either PtdIns(3)P or PtdIns(4)P (distinguished by labeling with DiI or DiD lipid dyes). Data were acquired at 1-s intervals for 150 s using three-color TIRF microscopy; each dot in the box plots represents the final uncoating efficiency for a single object. Box plots include the median, and data are from three independent experiments.