American Society of Hematology

Figure 2.

$Figure 2. Identification of components of the MeCPC purified from primary erythroid cells. (A) Chromatographic scheme (strategy I) used to purify the MeCPC from primary erythroid cells. The fractions eluted from each column were assayed for MeCPC activity by EMSA using the M-ρ248 probe. (B) EMSA on the eluted fractions from the final column of MeCPC purification strategy I (Heparin Sepharose HP) using the M-ρ248 probe. The MeCPC elutes in fractions 32 through 36. After 4 column chromatography steps the complex remains intact. (C, left) Sypro Ruby-stained protein gel containing 15 μg purified MeCPC. The identity of the bands was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprint data analysis. The molecular mass of markers in kDa is indicated. (Right) Identities of the bands in the gel. Proteins are grouped into columns based on the complex the protein has previously been associated with (MeCP1, eIF3, or Other). Four components of the MeCP1 complex were identified in the purified MeCPC sample: MBD2, RBAP48, HDAC2, and MTA1. Four components of the eIF3 complex as well as an associated protein were identified: eIF3i, eIF3h, eIF3e, eIF3d, and HSPC021. Five additional proteins were identified: β-actin, MENT, p50unk, BAF60b, and FMIP. The identity of the 2 smallest proteins in the gel could not be established.$

Identification of components of the MeCPC purified from primary erythroid cells. (A) Chromatographic scheme (strategy I) used to purify the MeCPC from primary erythroid cells. The fractions eluted from each column were assayed for MeCPC activity by EMSA using the M-ρ248 probe. (B) EMSA on the eluted fractions from the final column of MeCPC purification strategy I (Heparin Sepharose HP) using the M-ρ248 probe. The MeCPC elutes in fractions 32 through 36. After 4 column chromatography steps the complex remains intact. (C, left) Sypro Ruby-stained protein gel containing 15 μg purified MeCPC. The identity of the bands was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprint data analysis. The molecular mass of markers in kDa is indicated. (Right) Identities of the bands in the gel. Proteins are grouped into columns based on the complex the protein has previously been associated with (MeCP1, eIF3, or Other). Four components of the MeCP1 complex were identified in the purified MeCPC sample: MBD2, RBAP48, HDAC2, and MTA1. Four components of the eIF3 complex as well as an associated protein were identified: eIF3i, eIF3h, eIF3e, eIF3d, and HSPC021. Five additional proteins were identified: β-actin, MENT, p50unk, BAF60b, and FMIP. The identity of the 2 smallest proteins in the gel could not be established.

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