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Blood, Vol. 110, Issue 9, 3128-3135, November 1, 2007
Gfi1 ubiquitination and proteasomal degradation is inhibited by the ubiquitin ligase Triad1
Blood Marteijn et al.
110: 3128
Supplemental materials for: Marteijn et al
Files in this Data Supplement:
- Table S1. Primer sequences to generate Triad1 deletion mutants in sexual PCR (PDF, 48.8 KB) -
Primer  CC1+2R was used in regular PCR to introduce a stopcodon coding for Triad1 lacking the two C-terminal coiled coil domains.
- Figure S1. Triad1 inhibits Gfi1 ubiquitination (JPG, 10.6 KB)
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Cells were transfected as indicated. Triad1 inhibits poly-ubiquitination of Gfi1. Under these circumstances, the mono-ubiquitination of Gfi1 (arrowhead) was less affected by Triad1, indicating that Triad1 predominantly inhibits the poly-ubiquitination of Gfi1. (*) Marks non-specific interaction of Flag-Gfi1 with the beads.
- Figure S2. Triad1 siRNA1 and –2 downmodulate Triad1 in HEK293 cells (JPG, 17 KB)
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HEK293 cells were transfected with combinations of GFP-Triad1 with indicated siRNA constructs. 48 hours after transfection the mean fluorescence intensity (MFI) was measured by flow cytometer. The MFI of GFP-Triad1 transfected cells was set at 100%. Transfection with siRNA developed against Triad1 or GFP resulted in downregulated GFP-Triad1 protein levels, while the control siRNA showed no significant effect on GFP-Triad1 levels.
- Figure S3. Triad1 and Triad1dR2 expression after retroviral transduction (JPG, 7.25 KB)
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NIH3T3 cells were retrovirally transduced (transduction efficiency >95% based on NGFR expression measured by flowcytometer) with Triad1 and Triad1dR2 encoding viruses. Two days after transduction 400,000 cells were lysed and used in Western blotting and stained with an Triad1 antibody to demonstrate Triad1 and Triad1dR2 expression (samples were run on one and the same gel, the blot was scanned as a whole and non-relevant lanes were deleted).
- Figure S4. Gfi1 expression after viral transduction to U937 cells (JPG, 6.10 KB)
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U937 cells were retrovirally transduced with Gfi1 or control vector encoding viruses. 6 days after transduction when a total of 15% of cells were Gfi1 positive as based on GFP positivity, 100,000 cells were lysed and used in Western blot to confirm Gfi1 expression (samples were run on one and the same gel, the blot was scanned as a whole and non-relevant lanes were deleted).
- Figure S5. Atx-1 shows higher expression in granulocytes compared to monocytes (JPG, 19.2 KB)
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Relative Atx-1 mRNA expression in primary monocytes and granulocytes. Atx-1 is 4-fold higher expressed in granulocytes compared to monocytes. Atx-1 expression was measured using a pre-designed taqman gene expression assay and was GAPDH normalised. 3 paired samples of healthy volunteers were measured.
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