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Blood, Vol. 110, Issue 6, 2027-2033, September 15, 2007
Novel RUNX1 isoforms determine the fate of acute myeloid leukemia cells by controlling CD56 expression
Blood Gattenloehner et al.
110: 2027
Supplemental materials for: Gattenloehner et al, Vol 110, Issue 6, 2027-2033
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 192 KB)
- Table S1. FAB subtype and CD56 expression status of the ex vivo isolated acute myeloid leukaemia samples investigated in this study (PDF, 168 KB) -
n indicates number of cases
- Table S2. Commercial antibodies used in this study (PDF, 179 KB)
- Figure S1. (JPG, 36.1 KB)
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There was binding of the anti-exon 5.4 serum to the exon 5.4-expressing isoforms p30 and p24 but not to p48 and p38a. By contrast, the anti-RUNT domaine (anti-RUNX1 N-ter) antibody (sc-8563) bound to all isoforms, while the anti-RUNX1/exon 6 (anti-RUNX1 C-ter) antibody (sc-8564) exclusively binds to p48 and p38a.
- Figure S2. (JPG, 52.9 KB)
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The specificity of the anti-exon 5.4 antiserum was further tested by the following two experiments. (1) Competitive inhibition of binding of the anti RUNX1 Ex 5.4 antibody was demonstrated by adding increasing amounts of GST/RUNX1 Ex5.4 fusion protein as a competitor (and only GST protein as control, not shown). (2) Using heart tissue as a source of bona fide “ex vivo” natural RUNX1 proteins,2 concentration-dependent blocking of specific RUNX1 p30 detection was found, whereas nonspecific bands remained unaffected. NH indicates normal heart; ICM, ischemic cardiomyopathy.
- Figure S3. (JPG, 80.1 KB)
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To confirm that the anti-RUNX1 N-ter (sc-8563) antiserum does indeed recognize the aa 53-75 of RUNX1 (ie, the peptide that was supposedly used for immunization and production of sc-8563), the following peptide-mapping strategy was applied: overlapping 12-mer peptides (1-21), 7-mer peptides (22-46) and 10/11-mer peptides (47-53) (original scheme top panel), covering the aa 53-75 of RUNX1 were spotted on nitrocellulose (immunoglobe, Himmelstadt, Germany) (middle panel) and incubated with anti-RUNX1 N-ter (sc-8563) with subsequent chemilluminscent protein detection (bottem panel). Note identification of 7-mer peptide sequence LPLGAPD as specific recognition site in spot no. 29 as well as corresponding 12-mer spot nos. 3 through 8 and 10-mer spot no. 51. Moreover, the anti-RUNX1 N-ter (sc-8563) revealed specific antigen detection also in electro mobility shift assay (EMSA) demonstrated by super shift of DNA/RUNX1 protein complex (see supplement reference 2).
- Figure S4. (JPG, 87.8 KB)
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The new exon 5.4 (indicated as red bar in the above graph) is located on chromosome 21, q22.12, 35.091.532-35.092.261 and lies between the known exons 5.3 and 6 (see BLAST-like alignment tool (BLAT) search graphic below from University of California, Santa Cruz Genome Bioinformatics Group, Center for Biomolecular Science and Engineering, Santa Cruz, CA).
- Figure S5. Detection of the mRNA and protein expression of RUNX1 exon 5.4 by RNAse protection assay (above) and Western blot in normal human organs (JPG, 45.4 KB)
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(Top panel) Detection of RUNX1 exon 5.4 mRNA by RNAse protection assay in normal human tissues. Strongest and quite similar expression levels in heart (HE) and kidney (KI); weak-to-barely detectable expression in brain (BR), lymph node (LY), muscle (MU), spleen (SP), and thymus (TH). No detectable mRNA expression in duodenum (DD) and liver (LI). (Bottom panel) Western blot analysis of normal human organs with RUNX1 isoform-specific antibodies (see also Figure 1B in the print version of the article); anti-RUNX1 Runt-domaine (box 1), anti-RUNX1 exon 6 (box 2), anti-RUNX1 exon 5.4. (box 3), and anti-GAPDH (box 4, loading control).
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