Fig. 1: Brassinolide stabilizes PIN2:ubq:VEN.

a–d Comparison of eir1-4 PIN2p::PIN2:ubq:VEN root tips (a, b) and root epidermis cells (c, d) at 6 DAG germinated on PNS (a, c) or on PNS supplemented with 100 nM eBL (b, d). e PIN2:ubq:VEN signal ratios between PM and the intracellular space in PNS and eBL-treated seedlings. 82–85 root epidermal cells in 12 roots were tested for each dataset and analyzed by two-tailed t-test. f–i Western blots performed with eir1-4 PIN2p::PIN2:ubq:VEN membrane protein fractions isolated at 6 DAG probed with anti-PIN2; where indicated eir1-4 was used as a control. Anti-α-tubulin (TUB) was used as a loading control for all blots. f Samples either remained mock-treated (PNS) or were germinated in the presence of 100 nM 24-epiBL, or 28-homoBL. g Samples taken from a time course after 0, 90, and 360 min in the presence of 100 nM eBL ( + ) or incubated with solvent alone (-). h Samples with increasing concentrations of 24-epiBL. i Samples treated with 50 μM cholesterol (Chol.), 50 μM stigmasterol (Stig.), and 50 μM dexamethasone (DEX) for 16 h, or mock-treated sample (PNS). j PIN2 transcript levels in 6-day-old Col0 and PIN2p::PIN2:ubq:VEN on PNS or on 100 nM eBL for 6 h. Four biological repetitions were made for each sample, with transcripts normalized to expression of EF1a (At1g07940). k, l PIN2:ubq:VEN distribution and abundance in root epidermis cells of RP40p::PIN2:ubq:VEN at 6 DAG, on PNS (k) or in the presence of 100 nM eBL for 6 h (l). Whiskers in box plots cover the entire range of outliers obtained in the datasets; gray boxes: first and third quartiles; center line: median; dots: values obtained. Scale bars: a,b = 50 μm; c,d = 20 μm; k,l = 10 μm. Source data are provided as Source Data file.