Fig. 5: AFF1 interacts with ARF proteins and ASK1.
From: Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning
a GST, GST-AFF1 or GST-ΔF-box-AFF1 recombinant proteins were incubated with arf19-1 35 S:YFP-ARF19 plant lysate. Pull-down fractions and inputs were examined by immunoblot analysis using anti-GFP or anti-GST antibodies. b GST, GST-AFF1 or GST-ΔF-box-AFF1 were incubated with arf19-1 35 S:YFP-ARF19 plant lysate prior to immunoprecipitation with anti-GFP antibody. Immunoprecipitates and inputs were examined by immunoblot analysis using anti-GFP or anti-GST antibodies. c GST, GST-AFF1 or GST-ΔF-box-AFF1 were incubated with 35 S:ARF7-HA plant lysate prior to immunoprecipitation with anti-HA antibody. Immunoprecipitates and inputs were examined by immunoblot analysis using anti-HA or anti-GST antibodies. d GST, GST-AFF1 or GST-ΔF-box-AFF1 were incubated with His-ASK1 prior to pull down. Pull-down fractions and inputs were examined by immunoblot analysis using anti-His or anti-GST antibodies. e Bimolecular fluorescence complementation (BiFC; yellow) assays were used to analyze protein interactions between nEYFP-ΔF-box-AFF1 and cEYFP-ARF19, nEYFP-ΔF-box-AFF1 and cEYFP-IAA7, nEYFP-ARF19 and cEYFP-ARF19, or nEYFP-ARF19 and cEYFP-IAA7. The nuclear marker WPP-mCherry (magenta) was co-expressed to determine nuclear signal. Scale bar = 50 µm. See Supplementary Fig. 3 for extended data. f Left, confocal images of 3d-old wild type (Wt; Col-0) or aff1-1 seedlings carrying UBQ10:YFP-ARF19 or UBQ10:YFP-ARF19K962A (false-colored yellow) with cell walls counterstained with propidium iodide (false-colored magenta). Scale bar = 25 µm. Right, quantification of subcellular fluorescence. T (total), C (cytoplasmic), and N (nuclear). See Supplementary Fig. 4 for images from additional regions of the root. Three independent experiments were performed on (a), (b), (c), (d), (e) and (f) with similar results. n = 56 (ARF19) and n = 54 (ARF19K962A) independent cells were examined in (f). Data in (f) are mean ± SD and gray dots represent the individual values. Different letters indicate individual groups for multiple comparisons with significant differences (one-way ANOVA, Duncan, p  <  0.05). The source data in (a), (b), (c), (d) and (f) are provided as a Source Data file.
