[PubMed] [Google Scholar]Bernstein E, Caudy AA, Hammond SM, Hannon GJ. suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is usually a powerful approach to streamlining identification of protein partners. This value is usually then normalized by the sum of all these values across all proteins in the data set, correcting for differences in protein loading between data units. This result is usually then divided by the protein length, correcting for the propensity of longer proteins to produce a greater quantity of peptides than shorter proteins. Normalized spectral counts can then be used to provide ratios between sample and control for each protein (Griffin et al. 2010; Trudgian et al. 2011a). Protein candidates characterized by a minimum spectral count of five as well as a minimum enrichment ratio of 5:1 for sample versus control were identified as top candidates. Data are offered as normalized spectral counts for each significant protein. To increase statistical rigor when prioritizing protein ratings, we also applied Statistical Analysis of the Interactome (SAINT) analysis. Expression and purification of Flag-tagged and endogenous AGO2 In this study we used both Flag-tagged AGO2 and antibodies against endogenous AGO2 in parallel experiments. The advantage of using anti-AGO2 antibody is usually that it can detect endogenous AGO2 and its partners at normal levels of AGO2 expression. The disadvantage is usually that using anti-AGO2 antibody runs the risk of cross-reactivity with AGO1, AGO3, or AGO4 proteins. The use of Flag-tagged AGO2 is usually advantageous because the interaction between the Flag epitope and the antibody is usually selective and maximizes the potential to detect interacting factors. One disadvantage is usually that introducing a tag may alter the ability of AGO to interact with some proteins (Oeffinger 2012). A second potential disadvantage is usually that tagged AGO2 may not be expressed at the same level as endogenous AGO2. Identifying candidates that are pulled down after both strategies would streamline identification of candidate conversation partners for experimental validation. We used T47D cells because we had previously observed the involvement of AGO2 when promoter-targeted duplex RNAs were used to modulate gene transcription of the progesterone receptor in the nucleus of those cells (Janowski et al. 2005, 2007). We established T47D cells that stably express Flag-tagged AGO2. Western analysis of Flag-AGO2 cells using anti-AGO2 antibody suggests that AGO2 levels are approximately 4.7-fold higher than in the cytoplasm and 1.9-fold higher in the nucleus relative to endogenous AGO2 in T47D Mouse monoclonal to CD68. The CD68 antigen is a 37kD transmembrane protein that is posttranslationally glycosylated to give a protein of 87115kD. CD68 is specifically expressed by tissue macrophages, Langerhans cells and at low levels by dendritic cells. It could play a role in phagocytic activities of tissue macrophages, both in intracellular lysosomal metabolism and extracellular cellcell and cellpathogen interactions. It binds to tissue and organspecific lectins or selectins, allowing homing of macrophage subsets to particular sites. Rapid recirculation of CD68 from endosomes and lysosomes to the plasma membrane may allow macrophages to crawl over selectin bearing substrates or other cells. cells (Fig. 1B). The difference in expression of Flag-tagged and endogenous AGO2 underscores the value of including proteomic analysis of the endogenous protein. In T47D cells the levels of endogenous AGO2 were approximately equivalent in the nucleus and cytoplasm (Fig. 1C). In the Flag-AGO2 stable line, cells have a distribution of 67% cytoplasmic and 33% nuclear AGO2. Our laboratory (Gagnon et al. 2014a) as well as others (Schraivogel and Meister 2014) have previous visualized endogenous AGO2 in cell nuclei and we used Nalbuphine Hydrochloride microscopy to detect nuclear Flag-tagged AGO2 (Fig. 1D). Purification of nuclear or cytoplasmic AGO2 The previous studies identifying AGO2 interacting partners focused on preparations from whole cells, ribosomal gradient fractions, or chromatin (Meister et al. 2005; H?ck et al. 2007; Robb and Rana 2007; Landthaler et al. 2008; Weinmann et al. 2009; Ameyar-Zazoua et al. 2012; Frohn et al. 2012; Carissimi Nalbuphine Hydrochloride et al. 2015). One of our goals was to analyze cytoplasmic and nuclear interactions using extracts isolated from each compartment. Analysis of nuclear AGO2 requires demanding isolation of cell nuclei. AGO2 is found in the endoplasmic reticulum (ER), Nalbuphine Hydrochloride which is usually contiguous with the nuclear envelope (Stalder et al. 2013). We have previously developed protocols for separating nuclei from ER (Gagnon et al. 2014b). Western blot analysis exhibited that nuclear preparations lacked ER protein or cytoplasmic contaminants (Fig. 1E). Coomassie staining was routinely used as a quality control check to visualize an AGO2 protein band after immunoprecipitation to confirm adequate protection for Nalbuphine Hydrochloride mass spectrometry (Fig. 1F). Cytoplasmic interactions of AGO2 We began our analysis by examining the interactions of Flag-tagged AGO2 or endogenous AGO2 in isolated cytoplasmic extract from T47D cells (Fig. 2; Supplemental Furniture 1, 2). For initial experiments, RNase was not added so that RNA-mediated interactions remained intact. Mass spectrometry revealed potential interactions between Flag-tagged AGO2 and proteins from several different.