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. 2011 Sep 20;23(9):3463–3481. doi: 10.1105/tpc.111.086918

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© 2011 American Society of Plant Biologists. All rights reserved.

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Figure 5. — The ESCRT-I Component VPS28 Localizes to the Golgi and the TGN.

(A) Immunodetection of VPS28 in total protein extracts from 7-d-old Arabidopsis plants (left) using antibodies against VPS28 (αVPS28) and VPS28-GFP transiently expressed in protoplasts isolated from Arabidopsis suspension cultures (middle and right). Protoplasts were transfected with 3, 10, 30, or 100 μg plasmid DNA encoding for VPS28-GFP or mock transfected (−). Total protein extracts from protoplasts were probed with antibodies against VPS28 (αVPS28) and antibodies against GFP (αGFP).

(B) IEM analysis using the αVPS28 antibodies on high-pressure frozen Arabidopsis wild-type root cells shows that the endogenous VPS28 localizes to the Golgi stacks and the TGN (arrows).

(C) IEM of the endogenous VPS28 shows that VPS28 localizes to the Golgi stack and the TGN (arrows) but is not detected on the MVB

(D) Quantitative analysis of VPS28 IEM. The labeling density, expressed as the number of gold particles per micrometer² (gold/μm²), is significantly higher for the TGN and the Golgi apparatus (18.8 and 11.1 gold/μm², respectively) compared to the MVBs (3.1 gold/μm²) or plastids/mitocondria (1.4 gold/μm²). N°, number of compartments encountered; N° lab., number of compartments labeled; μm², total area considered; gold, total number of gold particles detected; gold/μm², labeling density.

(E) Double immunolocalization of VPS28 in an Arabidopsis line expressing the TGN marker SYP61-CFP under the control of the endogenous promoter, using the polyclonal αVPS28 antibodies from rabbit in combination with 15-nm (arrowheads) gold-coupled secondary antibodies and monoclonal αGFP antibodies from mouse in combination with 5-nm (arrows) gold-coupled secondary antibodies. Both, the TGN marker and VPS28 localize to the same tubular-vesicular structure, immediately adjacent to the Golgi stacks.

(F) In BFA-treated Arabidopsis plants, VPS28 labels the core of the BFA compartment, confirming TGN localization of this ESCRT-I subunit.

G, Golgi; T, TGN; M, MVB/LE; B, BFA compartment. Bars = 200 nm.