Extended Data Fig. 9: The nucleo-cytoplasmic partitioning of miRNAs and AGO1 in thp1-5.
From: Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis
a, Comparison of an improved nucleo-cytoplasmic fractionation method and the traditional method. The fractionated samples were subjected to protein gel blot analysis using anti-HYL1, anti-GAPDH and anti-H3 antibodies, respectively. T = total extract; C = cytoplasm; N = nucleus. HYL1 and H3 are nuclear proteins; GAPDH is a cytoplasmic protein. The experiment was repeated two times with similar results. b, Optimization of nucleo-cytoplasmic fractionation in terms of the duration of paraformaldehyde crosslinking (8min, 15min and 20min). The fractionated samples were subjected to protein gel blot analysis using anti-AGO1, anti-cFBPase, anti-HYL1, anti-BIP and anti-H3 antibodies, respectively. T = total extract; C = cytoplasm; N = nucleus. H3 is a nuclear marker; cFBPase is a cytoplasmic marker. They were also used in the quantification of AGO1 levels (represented by the numbers) between T and N and between T and C, respectively. Three independent experiments gave similar results. c, Small RNA gel blot assays to determine the levels of various miRNAs in Col and thp1-5 following fractionation with the improved method. T = total extract; C = cytoplasm; N = nucleus. Signal intensity of T was arbitrarily set to 1.0; that of C and N was normalized to T against U6 and tRNA-Met, respectively, as nuclear and cytoplasmic RNA markers. The experiment was repeated two times with similar results. d, Cytoplasmic/nuclear ratios of various miRNAs as determined in (c).
