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. 2019 Dec 4;10:5516. doi: 10.1038/s41467-019-13543-1

Fig. 3. Brassinolide antagonizes endocytic sorting of constitutively ubiquitylated PIN2.

Fig. 3

a 6 DAG eir1-4 PIN2p::PIN2:ubq:VEN on PNS, or treated with 100 nM eBL for 16 h, followed by 50 μM CHX or 100 nM eBL/50 μM CHX treatment for 30′, and by co-incubation for 60′ in the presence of 50 μM CHX/50 μM BFA and 100 nM eBL/50 μM CHX/50 μM BFA. White arrowheads: BFA-induced compartments. b 6-day-old eir1-4 PIN2p::PIN2:ubq:VEN grown on PNS or treated with 100 nM eBL for 16 h, followed by treatment with 50 μM CHX for 30′ (CHX), or co-treated with eBL and CHX for 30′ (eBL/CHX). This was followed by co-incubation in the presence of either 50 μM CHX and 30 μM WM (CHX/WM) or 100 nM eBL, 50 μM CHX and 30 μM WM (eBL/CHX/WM) for another 90′. White arrowheads: PIN2:ubq:VEN signals in WM-induced endocytic compartments; open arrowheads: PM localization. c PIN2:ubq:VEN signals in 6 DAG TOL and tolQ root epidermis cells, on PNS or on 10 μM BRZ for 16 h. White arrowheads: intracellular signals; open arrowheads: PM localization. d Frequency of BFA-compartment formation observed in ‘a' (n = 10–12 roots and 230-550 cells/dataset). e Signal ratios for ‘a' (n = 8 roots and 52/65 trichoblast cells/dataset). f Signal ratios for ‘b' (n = 5 roots and 32–33 cells/dataset). g Signal ratios for ‘c' (n = 4 roots and 34 cells/dataset). h Root meristem cells eir1-4 PIN2p::PIN2:ubq:VEN seedlings, on PNS (left, top); on 50 μM CHX for 5.5 h (top, right); on 100 nM eBL for 5 h (bottom, left); on 50 μM CHX for 30′, followed by eBL/CHX for 5 h (bottom, right). Open arrowheads: signals at the PM. i Signal ratios for ‘h’ (n = 4 roots and 20–26 cells/dataset). j eir1-4 PIN2p::PIN2:ubq:VEN root membrane fractions, probed for PIN2 and TUB (α-tubulin). Conditions as in ‘h’. Box plot whiskers represent the entire range of outliers; gray boxes: first and third quartiles; center line: median; dots: values obtained. Two-tailed t-test analysis has been performed to test for p-values. Scale bars: a–c, h = 10 μm. Source data are provided as a Source Data file.