Figure 6.

SlRd2 homodimerizes in the cytosol and in the nucleus. A, Y2H assay with both SlRd2 and SlRd2Δdim cloned in the bait pEG202 (with a LexA fusion tag at the C terminus) and prey pJG45 (with a HA fusion tag at the C terminus) vectors in EGY48 strain, grown in liquid culture and spotted at OD₆₀₀ = 0.1 and 0.01 on selective medium. Growth and blue patches indicates interaction. B, Expression of both SlRd2 and SlRd2Δdim proteins in yeast were confirmed by immunoblotting using anti-LexA (pEG202) and anti-HA (pJG45) antibodies, respectively. C, The cross-linking agent DSG was added to a His-SlRd2 overexpressing E. coli culture for 30 min. Polymerization of His-SlRd2 protein was calculated according to its molecular weight: 1×, monomer; and 2×, dimer. D, Colocalization analyses of SlRd2/SlRd2 complexes (yellow, a, d, and g) with the membrane marker dye FM4-64 (red, b), cytoplasmic marker AtFKBP12 (cyan, e), or nuclear marker DAPI (blue, h). Merged images are shown in (c), (f), and (i). Arrow denotes localization in the nucleus. Bars = 10 μm.