Anti-Sec21p | Gamma subunit, COP vesicles

GFP-OTU11 and GFP-OTU12 localize to the PM in Arabidopsis root cells.a Representative confocal images of 35S promoter-driven YFP-fusions of UBP3, UBP18, UBP22, UBP23, UBP25, UBP27, OTU9, OTU10, OTU11, and OTU12. YFP-fusion proteins were transiently expressed in Arabidopsis root cell-derived protoplasts. For each construct, protoplast transformation was performed at least twice. Scale bars: 10 µm. b Representative confocal images of protoplasts expressing 35pro:GFP-OTU11 or 35pro:GFP-OTU12 together with 35Spro:RFP-SYP121. The signal intensity profile along the dotted line (merged image) is shown on the right. Scale bars: 10 µm. Protoplast transformations were performed at least twice. c Representative confocal images of wild-type (Col-0) Arabidopsis seedlings harboring OTU11pro:GFP-OTU11 or OTU12pro:OTU12-GFP. Both lines were co-stained with FM4-64 for 5 min before imaging. The confocal analysis was performed at least twice. The signal intensity profile along the dotted line (merged image) is shown on the right of the panels. Scale bars: OTU11 10 µm, OTU12 20 µm. d GFP-OTU11 is present in the enriched PM fraction. Total proteins were prepared from wild-type (Col-0) seedlings containing 35Spro:GFP-OTU11. The extracts were centrifuged at 8000 ×g, and the supernatant (S8) was further centrifuged at 100,000 ×g to yield supernatant (S100) and microsome (P100) fractions. P100 samples were subsequently treated with Brij-58 for the enriched PM fraction (ePM). H+-ATPase, UGPase, and Sec21 were used as markers for the PM, soluble fraction, and microsomal fractions, respectively. Three independent experiments were carried out, and a representative result is shown. Source data are provided as a Source Data file. e, f35Spro:GFP-OTU11- and 35Spro:GFP-OTU12-expressing wild-type (Col-0) seedlings were stained with FM4-64 and treated with 50 µM brefeldin A (e) or 33 µM Wortmannin (f). The experiments were repeated at least three times; one representative image is shown. Scale bars: 10 µm. g35Spro:GFP-OTU11-, 35Spro:GFP-OTU12-expressing wild-type (Col-0) seedlings, and the PI(4)P-sensor (P5R)-containing seedlings were treated with 60 µM phenylarsine oxide (PAO). Whereas PAO treatment causes dissociation of P5R from the PM, localization of GFP-OTU11 and GFP-OTU12 remains unaffected. The experiment was repeated at least three times; one representative image is shown. Scale bars: 10 µm.

SYAC1 colocalizes with Golgi/TGN/endosomal/PVC markers.Co-imunolocalization of SYAC1-GFP with markers for different subcellular compartments (a) and quantification of colocalization using Pearson correlation coefficient (b). Five-day-old pEST:SYAC1-GFP seedlings grown on mock medium treated with 5 µM estradiol for 6 h used in co-imunolocalization experiments (n = 10 roots with 1–5 epidermal cells each). Co-imunolocalization performed using antibodies against anti-GFP and subcellular markers. In the boxplots, center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by Origin software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, individual data points are represented by dots. ER Endoplasmic reticulum, TGN trans-Golgi network, PVC prevacuolar compartment, PM plasma membrane. Scale bar 5 µm.