Fig. 3 C9ORF72 interacts with SMCR8 in a DENN domain–dependent manner.

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Fig. 3 C9ORF72 interacts with SMCR8 in a DENN domain–dependent manner. C9ORF72 interacts with SMCR8 in a DENN domain–dependent manner. (A and B) Flag-tagged SMCR8 or WDR41 was transfected into N2A cells. SMCR8 or WDR41 proteins were immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analysis using antibodies against endogenous C9ORF72. (C) Flag-tagged full-length or DENN domain–deleted SMCR8 was transfected into HEK293 cells. SMCR8 proteins were immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analysis using antibodies against C9ORF72 or WDR41. (D) Flag-tagged full-length or DENN domain–deleted C9ORF72 constructs were transfected into HEK293 cells. C9ORF72 proteins were immunoprecipitated with M2 beads (anti-Flag) followed by Western blot analysis using antibodies against SMCR8 or WDR41. (E) GFP-tagged C9ORF72 was cotransfected with red fluorescent protein (RFP)–tagged SMCR8 or WDR41 into N2A cells. Confocal micrographs of N2A cells stained with antibodies against GFP (C9ORF72; green) and RFP (SMCR8 or WDR41; red). Hoechst stains the nuclei (blue). Scale bars, 10 μm. (F) GFP-tagged DENN domain–depleted C9ORF72 was cotransfected with RFP-tagged SMCR8 into N2A cells (upper panels). GFP-tagged C9ORF72 was cotransfected with RFP-tagged DENN domain–depleted SMCR8 into N2A cells (lower panels). Confocal micrographs of N2A cells stained with antibodies against GFP (C9ORF72ΔDENN or C9ORF72; green) and RFP (SMCR8 or SMCR8ΔDENN; red). Hoechst stains the nuclei (blue). Scale bars, 10 μm. Mei Yang et al. Sci Adv 2016;2:e1601167 Copyright © 2016, The Authors