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. 2020 Mar 19;32(5):1644–1664. doi: 10.1105/tpc.19.00869

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Figure 8. — ALA3 Physically Interacts with ARF GEFs.

(A) and (B) BiFC analysis of the interactions of ALA3 (A) or ALA3^G234E (B) with ALIS1, with GNOM and with BIG3 in N. benthamiana leaf epidermal cells. The split nYFP fusions nYFP-ALA3 or nYFP-ALA3^G234E were co-expressed with cYFP-ALIS1, or with cYFP-GNOM and untagged ALIS1, or with cYFP-BIG3 and untagged ALIS1 in N. benthamiana leaf epidermal cells. YFP signals were checked at ∼72 h after infiltration. The images were collected with the same settings used for confocal microscopy, including the laser power, pinhole size, and gain level. The experiment was repeated three times with similar results. Scale bars = 100 μm.

(C) Co-IP assays testing the interactions of ALA3 with GNOM or BIG3 in N. benthamiana. GNOM-FLAG or BIG3-FLAG was transiently co-expressed with HA-ALA3 and CFP-ALIS1 in N. benthamiana leaf epidermal cells. Plants transiently expressing BIG3-FLAG were used as a positive control, and plants transiently co-expressing HA-ALA3 and CFP-ALIS1 were used as a negative control. Proteins were extracted from infiltrated leaves and analyzed using anti-FLAG and anti-HA antibodies. Input levels of HA-ALA3 protein in the crude protein extracts were analyzed by immunoblotting with anti-HA antibody. Immunoprecipitated (IP) BIG3-FLAG or GNOM-FLAG proteins were probed with anti-HA antibody to detect Co-IP of HA-ALA3.