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. 2020 Aug 4;32(10):3290–3310. doi: 10.1105/tpc.20.00267

Figure 6.

Figure 6.

SINAT Proteins Form Homo- and Hetero-Oligomers in Vivo.

(A) Y2H assay showing mutual interactions between SINAT proteins. Mutated SINAT proteins SINAT1-C60S, SINAT2-C63S, SINAT3-C66S, SINAT4-C67S, SINAT5, and SINAT6 were fused with AD or BD and then paired into a 6 × 6 array for the interaction assay. Three truncated forms of SINAT1 (SINAT1-RZ, SINAT1-ZT, and SINAT1-T) were also fused with BD and cotransformed with six AD-SINATs to identify the binding domain. The plasmids were coexpressed in the YH109 yeast strain and selected on SD-Trp-Leu-His-Ade medium (SD-LWH-Ade). AD and BD indicate the empty AD and BD plasmids, respectively. R, RING finger; T, TRAF domain; Z, ZINC finger.

(B) Co-IP assay for physical interaction of SINAT1 with six SINAT proteins (SINAT1, SINAT2, SINAT3, SINAT4, SINAT5, and SINAT6). Plasmids of FLAG-tagged SINAT1, SINAT2, SINAT3, SINAT4, SINAT5, SINAT6, or empty vector (EV) were transfected into Arabidopsis protoplasts isolated from a GFP-SINAT1-HA transgenic line. Protoplasts were cultured overnight and were then lysed with IP buffer and incubated with anti-FLAG magnetic beads.

(C) BiFC assay for the association between SINAT proteins. nYFP-fused SINAT1 or SINAT2 was coexpressed with cYFP-fused SINAT1, SINAT1-RZ, or SINAT1-ZT in the wild-type (WT) Arabidopsis protoplasts. Signals were detected after overnight culture by confocal microscopy. The reconstituted YFP signal suggests an interaction between two proteins. Bars = 10 μm.

(D) Oligomer formation of SINAT proteins in vivo. Total proteins were extracted from 1-week-old GFP-SINAT1-HA, GFP-SINAT2-HA, SINAT3-GFP-HA, and GFP-SINAT4-HA transgenic seedlings with IP buffer and were denatured with SDS sample buffer with or without 1% 2-ME. SINAT proteins were detected with anti-HA antibodies. A Ponceau-stained membrane shown below the blot indicates the loading protein amounts. The relative intensities of oligomers and monomers normalized to ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) are shown below, with each 2-ME–containing samples set as 1. Double asterisk (**) indicates oligomers of SINAT proteins. Numbers on the left indicate the molecular weight (kD) of each band.