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. Author manuscript; available in PMC: 2021 Jan 20.
Published in final edited form as: Nat Plants. 2020 Jul 20;6(8):957–969. doi: 10.1038/s41477-020-0726-z

Extended Data Fig. 9. The nucleo-cytoplasmic partitioning of miRNAs and AGO1 in thp1–5.

Extended Data Fig. 9

(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).