American Society of Hematology

Figure 3.

$Figure 3. Putative components of the MeCPC copurify from a Superose 6 gel-filtration column. (A) The second chromatographic scheme (strategy II) was used to purify the MeCPC from primary erythroid cells. The fractions eluted from each column were assayed for MeCPC activity by EMSA using the M-ρ248 probe. (B) EMSA on the high molecular weight fractions from the final column of MeCPC purification strategy II (Superose 6) using the methylated M-ρ248 probe. The MeCPC elutes primarily in fractions 8 through 15 in a molecular weight range of 670 to 2000 kDa. The position of the MeCPC is indicated as “M,” the slower-migrating supercomplex as “Sup,” and the faster-migrating subcomplex as “Sub.” Although the exact composition of the supercomplex is unknown, we hypothesize that the subcomplex results from cleavage of MeCPC component proteins during biochemical purification. (C) EMSA on the high molecular weight fractions from the final column of MeCPC purification strategy II (Superose 6) using the unmethylated ρ248 probe. Both the MeCPC and the supercomplex fail to retard the mobility of an unmethylated probe. In contrast, the subcomplex is able to retard the ρ248 probe (fractions 18 and 19). (D) Western blot analysis of the high molecular weight fractions from the Superose 6 gel-filtration column. The antibody used for each blot is indicated at the right. The putative MeCPC components MBD2, RBAP48, HDAC2, p66, MTA1, and Mi2 copurify from the column. In addition, the erythroid-specific heterochromatin protein MENT also copurifies with the MeCPC factors.$

Putative components of the MeCPC copurify from a Superose 6 gel-filtration column. (A) The second chromatographic scheme (strategy II) was 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 high molecular weight fractions from the final column of MeCPC purification strategy II (Superose 6) using the methylated M-ρ248 probe. The MeCPC elutes primarily in fractions 8 through 15 in a molecular weight range of 670 to 2000 kDa. The position of the MeCPC is indicated as “M,” the slower-migrating supercomplex as “Sup,” and the faster-migrating subcomplex as “Sub.” Although the exact composition of the supercomplex is unknown, we hypothesize that the subcomplex results from cleavage of MeCPC component proteins during biochemical purification. (C) EMSA on the high molecular weight fractions from the final column of MeCPC purification strategy II (Superose 6) using the unmethylated ρ248 probe. Both the MeCPC and the supercomplex fail to retard the mobility of an unmethylated probe. In contrast, the subcomplex is able to retard the ρ248 probe (fractions 18 and 19). (D) Western blot analysis of the high molecular weight fractions from the Superose 6 gel-filtration column. The antibody used for each blot is indicated at the right. The putative MeCPC components MBD2, RBAP48, HDAC2, p66, MTA1, and Mi2 copurify from the column. In addition, the erythroid-specific heterochromatin protein MENT also copurifies with the MeCPC factors.

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