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. 2017 Aug 7;114(34):E7197–E7204. doi: 10.1073/pnas.1710866114

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Fig. 5. — DUB AMSH3 interacts directly with SH3P2 and VPS23.1 and localizes on both CLC- and ARA7-positive endosomes. (A) In vitro-binding assay of SH3P2 with AMSH3. Bead-bound GST-AMSH3 and the negative control GST-VPS2.1 were incubated with equal amounts of full-length MBP-SH3P2. After intensive washing, bead-bound materials were analyzed by immunoblotting with anti-MBP and anti-GST antibodies. (B) YTH analysis of AMSH3 with ESCRT-I subunits. GAD-AMSH3 or GBD-AMSH3(AXA) was coexpressed with ESCRT-I subunits GBD-VPS28.1, GBD-VPS37.1, and GAD-VPS23.1. Yeast transformants expressing both GAD- and GBD-fusions were grown on media lacking leucine and tryptophan (−LW) or leucine, tryptophan, and histidine (−LWH) to test their auxotrophic growth. Empty vectors were used as negative controls. The expression of fusion proteins was verified by immunoblotting, as shown in Fig. S5. (C) In vitro-binding assay of AMSH3 with VPS23.1. Bead-bound GST-VPS23.1 was incubated with an equal amount of AMSH3. After intensive washing, bead-bound materials were analyzed by immunoblotting with anti-AMSH3 and anti-GST antibodies. The arrowhead indicates the position of GST-VPS23.1. (D) AMSH3 coimmunoprecipitates with VPS23.1-HA. Total extracts of wild-type (WT) and VPS23.1-HA seedlings were incubated with anti-HA antibody-immobilized agarose. After intensive washing, the immunoprecipitated (IP) material (anti-HA) was subjected to immunoblotting together with the total extracts (input) using anti-HA and anti-AMSH3 antibodies. (E) Colocalization of AMSH3-YFP and CLC-mKO. The localization of AMSH3-YFP and CLC-mKO in root epidermis cells of 7-d-old seedlings was analyzed under a confocal microscope. (Scale bar: 10 μm.) (F) Colocalization of AMSH3-YFP with CLC-mKO and ECFP-ARA7. The localization was analyzed under a confocal microscope in root epidermis cells of 7-d-old seedlings expressing AMSH3-YFP with CLC-mKO and ECFP-ARA7 without (Left) or with 60 min of WM treatment (Right). (Scale bars: 10 μm.) (G) Percentage of AMSH3-YFP foci that colocalize with CLC only, ARA7 only, with both CLC and ARA7, or with neither marker (without WM treatment). n, total number of analyzed AMSH3-YFP foci.