Fig. 1
Direct protein–protein interaction among ECT2, ECT3, and ECT4. a BiFC assay showing the physical associations among ECT2, ECT3, and ECT4 in Nicotiana benthamiana leaf cells. The association of paired proteins is indicated by YFP fluorescence in the cytoplasm. Scale bars = 40 μm. b Y2H assay showing the physical associations among ECT2, ECT3, and ECT4 in yeast cells. The full-length coding sequences of ECT2, ECT3, and ECT4 were fused with either the GAL4-AD or BD domain as indicated. YSD-Leu-Trp-Ade-His, selective medium without tryptophan, leucine, histidine, or adenine; YSD-Leu-Trp, medium without tryptophan or leucine (growth control). c Pull-down assay showing the interaction among GST-ECT2, MBP-ECT3, and SUMO-ECT4 in vitro. Purified MBP-ECT3 and SUMO-ECT4 proteins were incubated with GST-ECT2 or GST alone, and pull-down assays were performed using GST magnetic beads, followed by immunoblot analysis with anti-GST, anti-MBP, and anti-SUMO antibodies. d Correlation analysis of mRNA expression levels in Arabidopsis among ECT2, ECT3, and ECT4 in the ATTED-II database (n > 10,000 samples; ρ, Spearman’s correlation coefficient). P-values were calculated with Pearson’s correlation analysis. e The protein level of ECT2–FLAG in indicated samples, as determined by western blot. β-Actin protein was used as the loading control. f In vivo FA-RIP assay showing that ECT2-IP RNA amount and m6A level is enriched in ECT2:ECT2/ect2-1 compared with ECT2:ECT2/ect2/3/4 plant. IgG-IP was used as control. Data are presented as means ± SE, n = 3 biological replicates × 2 technical replicates. *P < 0.05, **P < 0.01 (two-sided t-test)