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Published Erratum
. 2015 May 12;112(19):E2554.
doi: 10.1073/pnas.1506636112. Epub 2015 Apr 16.

Correction for Brodersen et al., Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis

No authors listed
Published Erratum

Correction for Brodersen et al., Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis

No authors listed. Proc Natl Acad Sci U S A. .
No abstract available

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Figures

Fig. 5.
Fig. 5.
AGO1 is a peripheral membrane protein. (A) Western analysis of AGO1, HMG1, and PEPC proteins in 100,000 × g supernatant (sup) and pellet (pel) fractions of cleared Col-0 inflorescence extracts prepared by hypertonic lysis. Five percent of the supernatant fraction is loaded, 20% of pellet fraction is loaded, precluding any clear estimates of relative abundance in soluble versus insoluble fractions. HMG1 is used here solely as a positive control for a transmembrane protein. (B) Western analysis of AGO1 and HMG1 proteins in pellet fractions prepared as in A. Pellets were resuspended in either microsome buffer, or microsome buffer supplemented with 1 M KCl, 0.1 M Na2CO3, or 0.5% Triton X-100, and separated into supernatant (sup) and pellet (pel) fractions again at 100,000 × g. At longer exposures, an insoluble AGO1 fraction upon Triton X-100 treatment is visible. (C) Microsome fractionation of inflorescence lysates from Col-0, ago1-25, and ago1-38. Two experiments with corresponding total (tot.) and microsome (micr.) fractions analyzed by Western blots with AGO1 antibodies are shown. (Top two panels) Western blots developed by enhanced chemiluminescence. (Bottom two panels) Western blots developed by less sensitive alkaline phosphatase staining that allows a clearer visualization of the difference in microsomal AGO1 abundance between Col-0 (WT) and ago1-38. *Unspecific cross-reacting band; RbcL, large subunit of RuBisCO; BSA, BSA added to the microsome buffer to avoid membrane association of proteins post lysis. The amount of BSA recovered in washed pellets is proportional to the amount of membrane recovered, and is used as loading control. (D) (Upper) Schematic depicting the position of several AGO1 hypomorphic mutations used in this study (black triangle) and the ago1-38 allele (red triangle) causing the G186R missense mutation. (Lower) The glycine residue mutated in Arabidopsis ago1-38 is highly conserved among various metazoan and fungal AGO proteins (highlighted in yellow). At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Sp, Schizosaccharomyces pombe. (E) Microsome fractionation of inflorescence lysates from Col-0 and hmg1-3. (Upper) Western analysis of AGO1 in total extracts. (Lower) Western analysis of AGO1 and HMG1 in microsome (pel) fraction. (F) Same analysis as in E performed with GFP171.1 and mad3 inflorescence lysates.
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