PLANT BIOLOGY Correction for “Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis,” by Peter Brodersen, Lali Sakvarelidze-Achard, Hubert Schaller, Mehdi Khafif, Grégory Schott, Abdelhafid Bendahmane, and Olivier Voinnet, which appeared in issue 5, January 31, 2012, of Proc Natl Acad Sci USA (109:1778–1783; first published January 12, 2012; 10.1073/pnas.1112500109).
The authors wish to note, “It has been brought to our attention that a loading control panel in Fig. 5C had been incorrectly assembled. We have verified that mistakes were indeed made in assembling the loading control panels in the upper half of Fig. 5C. We have retrieved the original exposures of the Western blots and Coomassie-stained membranes. The original data show that the conclusion drawn on the basis of the data in Fig. 5C, that in inflorescences, ago1-38 mutant protein is less abundant specifically in microsomal fractions, remains valid. The corrected figure and updated figure legend appear below.”
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.