Fig. 5: amiRCWC15 affects the degradation of SE.
From: The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis
a The protein levels of DCL1, HYL1, SE detected by western blot in Col and amiRCWC15. Rubisco stained with Coomassie brilliant blue and HSC70 were used as loading control.DCL1 was detected by anti-DCL1(1: 1000 dilutions, rabbit polyclonal, AS194307, Agrisera). Three independent experiments yielded consistent results. b SE levels in Col and amiRCWC15 seedlings with or without MG132 treatment determined by western blot. Rubisco serve as loading control. Two independent experiments yielded consistent results. c In vitro cell-free SE-decay assay. Total proteins from Col and amiRCWC15 were extracted and incubated with CHX (0.5 mM) and ATP (5 mM) for the indicated times. SE levels were determined by western blot. Rubisco and ACTIN2 serve as loading control. Two independent experiments yielded consistent results. d PAG1 IPed from 10 day-old plants detected by western blot using an anti-FLAG antibody. IPed PAG1-FM was eluted with the FLAG peptide. Two independent experiments yielded consistent results. e Silver-staining of IPed PAG1-FM complex resolved in 10% SDS-PAGE. The arrow and bracket indicate PAG1-FM proteins and subunits of the 20 S core proteasome (CP), respectively. Two independent experiments yielded consistent results. f In vitro reconstitution assays of SE degradation via affinity purified 20 S proteasome. Recombinant SE proteins were incubated with IPed PAG1-FM complex from Col or amiRCWC15 harboring pPAG1::gPAG1-FM. The reaction mixture was stopped at the indicated time intervals. The numbers below the gels indicate the relative mean signals of SE proteins at different time points that were normalized to those of the proteins at time 0, where the value was arbitrarily assigned a value of 1. g Cell-fractionation analysis of SE protein from Col and amiRCWC15 plants. Total extraction (T), nuclear fraction (N) and cytoplasmic fraction (C). Western blot analysis was conducted with an anti-SE or anti-HYL1 antibodies. Rubisco stained with Coomassie brilliant blue and histone 3 detected by anti-H3 antibody were used as controls for the cytoplasmic- and nuclear-specific fractions, respectively. h Quantification of the nuclear–cytoplasmic distribution of SE protein. The nuclear and cytoplasmic fraction ratios (N/C) of SE protein in amiRCWC15 were sequentially normalized to that of Col-0, which was arbitrarily assigned a value of 1. Bar graph representing the relative N/C ratios derived from band intensity. Error bars indicate SD between two biological replicates (n = 2) with two-tailed unpaired t-test, p-values indicated above plot. Source data are provided as a Source Data file.
