JCS016865 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Salt elution profiles of EGFP tagged MECP2e1 and MECP2e2. (A) Nuclei were isolated from stable cell lines expressing EGFP-tagged MECP2e1 and MECP2e2. Nuclear proteins were extracted at increasing salt concentrations. The supernatant consists of MECP2 and other nuclear proteins eluted at a given salt concentration. MECP2 remaining bound to the chromatin in the pellet was extracted with 1 M NaCl. The ratio of supernatant:pellet indicated the amount of MECP2 released at a particular salt concentration. The proteins were separated on a 7.5% SDS-PAGE and immunoblotted with an antibody generated against the C-terminus of MeCP2. Both endogenous and EGFP-tagged MECP2e1 and MECP2e2 exhibited very similar salt elution profiles. S, supernatant; P, pellet. (B) The MECP2e1 blot in A was quantified and graphed. The y-axis represents the percentage of protein remaining bound at a particular salt concentration. (C) MECP2e2 was quantified using NIH ImageJ. The y-axis represents the percentage of protein remaining bound at a particular salt concentration.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Basal expression of MECP2 or its mutants does not alter chromatin structure. Nuclei were isolated from the indicated cell lines and digested with or without micrococcal nuclease (Mnase). Total DNA (digested and undigested) was isolated resolved on a 1.8% metaphor agarose gel and stained with ethidium bromide. 3T3 represents the untransfected cell line.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Basal protein expression of the different mutant cell lines. Whole-cell lysates were prepared from stable cell lines expressing the isoform-specific and domain-deletion constructs. Untransfected cells are represented in the 3T3 lane. The proteins were resolved on a 4-15% gradient SDS-PAGE and immunoblotted. The resultant bands were quantified using the ImageJ software.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Localization of the R168X mutant protein. Cells expressing WT or the RTT truncation mutation R168X were imaged by confocal microscopy. The WT protein is exclusively nuclear, whereas R168X accumulates both in the nucleus and cytoplasm. Scale bar, 5 µm.