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Blood, Vol. 112, Issue 9, 3777-3787, November 1, 2008
PEBP2-β/CBF-β–dependent phosphorylation of RUNX1 and p300 by HIPK2: implications for leukemogenesis
Blood Wee et al.
112: 3777
Supplemental materials for: Wee et al
Files in this Data Supplement:
- Figure S1. Mapping of region in PEBP2β responsible for the mediation of RUNX1 phosphorylation (JPG, 36.8 KB)
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HEK293 cells were transfected with RUNX1 (100ng) and the indicated untagged PEBP2β expression vectors (300ng). The cells were harvested after 24h and analysed by Western blotting to determine the phosphorylation status of RUNX1 by PAGE electrophoretic mobility. PEBP2β1, β2, and β3 are naturally occurring isoforms of the PEBP2β subunit. The β117 deletant is unable to dimerize with RUNX1. Expression of PEBP2β is confirmed by Western blotting using anti PEBP2β antibody. Due to the deletion of the targeted epitope, this antibody cannot detect the β117 deletant.
- Figure S2. The SRC -interacting domain (SID) and bromodomain of p300 are necessary for RUNX1/PEBP2β-induced p300 phosphorylation (JPG, 22 KB)
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Deletions of p300 domains (Figure 4A) were performed employing PCR-based methodologies as described in Materials and Methods, targeting residues: 328430 for ΔCH1; 567663 for ΔKIX; 17301808 for ΔCH3; and 19792139 for ΔSID. HEK293 cells were co-transfected with RUNX1 and PEBP2β and FLAG-p300 mutants bearing deletion in CH1, KIX, CH3, SID or bromo- domains. Cells are lysed after 24 h and subjected to SDS-PAGE and Western blotting with anti-FLAG (top panel), anti-RUNX1 (middle panel) and anti-PEBP2β (bottom panel) antibodies. The involvement of bromodomain suggests the necessity of chromatin association for phosphorylation, and indicates direct interaction between HIPK2 and the p300/RUNX1/PEBP2β ternary complex.
- Figure S3. Generation of a phospho-specific anti-RUNX1 antibody (JPG, 31.3 KB)
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A polyclonal antibody specific for RUNX1 phosphorylated at Thr273 and Ser276 was generated by BioGenes Co. (Berlin, Germany). Rabbits were immunized with a phosphorylated peptide, VHPA(pT)PI(pS)PGR, which mimics phosphorylated RUNX1. The collected sera were first depleted for antibodies (αnonP-RUNX1) specific for non-phosphorylated form of the peptide (VHPATPISPGR), before affinity-purified using columns with the phosphorylated peptide. Figure S1 shows that this antibody recognised phosphorylated RUNX1 exclusively, unlike the antibodies targeting the Runt domain or unphosphorylated RUNX1.
- Figure S4. Leukemogenic fusion proteins disrupt RUNX1/PEBP2β-mediated p300 phosphorylation (JPG, 38 KB)
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FLAG-p300 was expressed alone or with PEBP2β-SMMHC and RUNX1-ETO in the presence of RUNX1 or PEBP2β proteins, respectively. Total cell lysates were separated by SDS-PAGE and probed with the anti-RUNX1 and -PEBP2β antibodies (top panel), or anti-FLAG antibody (bottom panel). This blot is a composite image from a single exposure with some lanes deleted. Together with uneven electrophoresis during SDS-PAGE, this might have resulted in some misalignment of bands.
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