| In Vitro |
ES9-17 is a CME inhibitor a, ES9-17 inhibited recruitment of the plasma membrane-localized BRI1-GFP to the BFA body. Samples were pretreated with 50?��M CHX for 1?h and treated with either DMSO (indicated as ?) or ES9-17 (30?��M) for 30?min, followed by a combined application of FM4-64 (2?��M) and BFA (50?��M) for an additional 30?min in the presence of the inhibitor. Scale bar, 10?��m. b, Confocal images of Arabidopsis root epidermal cells expressing PEPR1-GFP. Seedlings were treated with ES9-17 (30?��M) or DMSO (?) for 30?min, followed by elicitation with 100?nM Pep-1. The internalization of PEPR1-GFP was followed until 90?min post elicitation. Scale bar, 20?��m. c, Histograms representing the measured endocytic foci lifespan of CLC1-GFP (CLC1:CLC1-GFP/Col-0) in the presence of ES9-17 (30?��M) or DMSO (?); n?=?1,276 and 1,069 measurements from ten and four seedlings, respectively. Kymographs represent a line trace (horizontal axis), over a time period (vertical axis) taken from a time lapse (2?frames?s�C1) of Arabidopsis root epidermal cells that illustrates the lifespan of endocytic foci labeled by CHC1-GFP. Scale bar, 10?s. The experiments in a�Cc were repeated independently three times with similar results. The life time distribution graph was generated by grouping the events into specific categories, annotated as, for example, 0 �� x < 10: the category holding those events with a life time (x) being larger or equal (��) than 0 s and smaller (<) than 10 s.
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