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Blood, Vol. 108, Issue 8, 2836-2845, October 15, 2006
MBD2 is a critical component of a methyl cytosine-binding protein complex isolated from primary erythroid cells
Blood Kransdorf et al.
108: 2836
Supplemental materials for. Kransdorf et al
Files in this Data Supplement:
- Table S1. Primer sequences for Q-PCR (PDF, 89 KB)
- Table S2. Characteristics of proteins in the MeCPC sample identified by MALDI-TOF mass spectrometry (PDF, 118 KB)
- Figure S1. Identification of the chicken homolog of MBD2 (JPG, 112 KB)
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Comparison of the amino acid sequences of chicken, mouse, and human MBD2. Chicken MBD2 is 83% identical to human MBD2 over an overlapping 241 amino acid region (underlined above) that corresponds to the MBD2b isoform found in mammalian cells. Translation initiation sites are shown in bold. The cDNA sequence for chicken MBD2 has been deposited in Genbank as accession number AY888006.
- Figure S2. Antibodies prepared against cMBD2 recognize a protein at 28 kD in multiple chicken cell types (JPG, 30.3 KB)
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A. Western blot with anti-cMBD2 antibody V1 on HeLa nuclear extract, recombinant GST/cMBD2, chicken embryonic fibroblast (CEF) nuclear extract, chicken pre-B cell (DT40) nuclear extract, and chicken RBC extract. The antibodies, prepared against a recombinant protein produced from the cMBD2 cDNA, recognize a protein of 28 kDa in the chicken cell lines.
B. Western blot with anti-cMBD2 antibody V2 with samples as for A. The band at 28 kDa was specifically abolished by incubating the antibody with an excess of recombinant cMBD2.
- Figure S3. mMBD2 and MTA2 bind to the methylated ρ-globin gene in MEL cells containing a stably integrated ρ-globin mini-locus (JPG, 46.1 KB)
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A.EMSA performed with 20 μg of MEL cell nuclear extract shows that the MeCPC is also present in this mouse erythroid cell line. The addition of anti-mMBD2 IgG, but not control IgG, retarded the mobility of the MeCPC complex (supershift) during EMSA.
B. Enrichment for mMBD2 and MTA2 at the  -globin gene in mMBD2+ MEL- (grey) and mMBD2-knock-down MEL- (black) cells as determined by ChIP assay. Because chromatin immunoprecipitated with control (non-specific) anti-sheep and anti-goat IgG showed slight enrichment for -globin sequences, we used the measure of “net enrichment” to determine protein occupancy in chromatin. After chromatin immunoprecipitation with anti-mMBD2, anti-MTA2, or control antibodies, the enrichment for -globin sequences (bound/input) was normalized to the enrichment for the inactive amylase gene. To calculate net enrichment, the -globin/amylase ratio obtained from chromatin precipitate using a control antibody (non-specific IgG) was subtracted from the -globin/amylase ratio obtained from chromatin precipitate using a specific antibody (anti-mMBD2 or anti-MTA2). MEL- cells treated with shRNAs targeting mMBD2, in which mMBD2 expression is knocked-down by greater than 90%, were used as a control. The data show that mMBD2 and MTA2 are highly enriched at the transcriptionally inactive and methylated -globin gene in mMBD2+ MEL- cells. In contrast, no enrichment for mMBD2 or MTA2 is seen at the transcriptionally active -globin gene in mMBD2-knock-down MEL- cells. The data represent the average of three independent experiments, with the calculated standard deviation indicated by the bar.
- Figure S4. mMBD2 expression is lost in MEL cells treated with shRNAs targeting mMBD2 (JPG, 30 KB)
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A. Western blot with anti-mMBD2 antibody on MEL- cells. Cells expressing shRNAs lack mMBD2 expression (left lane), whereas cells containing empty vector still express mMBD2 (right lane).
B. Western blot with anti- -actin antibody on MEL- cells. Cells expressing shRNAs, (left lane) as well as cells containing empty vector (right lane), have equal -actin expression. This verifies that equal amounts of nuclear protein were loaded to the gel.
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