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. Author manuscript; available in PMC: 2018 Feb 23.
Published in final edited form as: Sci Transl Med. 2017 Aug 23;9(404):eaaf7588. doi: 10.1126/scitranslmed.aaf7588

Fig. 1.

Fig. 1

Screening for post-translational regulators of MeCP2. (A) The bicistronic reporter transgene allows for monitoring of MeCP2 stability via C-terminal fusion of EGFP, while controlling for variation in transgene expression by normalization to the independently translated DsRed. (B) Flow cytometry analysis of a clonal Daoy DsRed-IRES-hMECP2-EGFP reporter cell line, indicating dual fluorescence and minimal expression variation. (C) Schematic for arrayed siRNA screen against all known and putative human kinases and phosphatases by flow cytometry analysis of the MeCP2-GFP/DsRed ratio. (D) 181 siRNAs significantly (p <0.01) altered the MeCP2-EGFP/DsRed ratio in the primary screen against human phosphatases (711 siRNAs total). (E) 299 siRNAs significantly (p <0.01) altered the MeCP2-EGFP/DsRed ratio in the primary screen against human kinases (1908 siRNAs total). (F) A secondary screen using independent siRNAs confirmed 120 significant siRNAs affecting the MeCP2-EGFP/DsRed ratio. siRNAs that also significantly altered a GFP control cell line were eliminated, revealing 43 MeCP2-specific siRNAs, representing 33 genes.