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Blood, Vol. 110, Issue 7, 2371-2380, October 1, 2007
Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia
Blood Kile et al.
110: 2371
Supplemental materials for Kile et al, Vol. 110, Issue 7, 2371-2380
Files in this Data Supplement:
- Figure S1. Wild-type mRNA levels are decreased in mice carrying the rd allele of Wdr1 (PDF, 98.6 KB) -
Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of Wdr1 mRNA in adult tissues and CD41 positive cells sorted from bone marrow. A Taqman minor groove binder probe specific for the sequence deleted in the mutant transcript produced by cryptic splicing of the rd allele specifically detected wild-type message. Data represent mean plus or minus SD, 4 mice per genotype. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
- Figure S2. The mutant form of Wdr1 is unstable and potentially misfolded (PDF, 325 KB) -
(A) Pulse chase experiments. Plasmids encoding flag-tagged wild-type (i) or mutant forms of Wdr1 produced by cryptic splicing at the rd allele (Wdr1Δ346-347) (ii) were transfected into 293T cells, which were then pulsed with S35, and chased with cold media for the indicated time periods. Cells were lysed, immunoprecipitated with anti-Flag antibodies, and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Phosphoimaging (top panels) revealed that Wdr1Δ346-347 had a significantly reduced half-life. (B) Immunoprecipitation (IP) of transiently transfected 293T cells showed that a mouse anti-Wdr1 monoclonal antibody was capable of recognizing both wild-type and Wdr1Δ346-347 by western blot, but only the wild-type by IP, suggesting that Wdr1Δ346-347 is misfolded.
- Figure S3. A gene trap insertion at the Wdr1 locus (PDF, 337 KB) -
(A) The XN462 ES cell line contains a gene trap insertion in intron 2 of Wdr1 (B) Male mice that are compound heterozygous for the rd and the gene trap allele of Wdr1 are runted relative to littermates (i) and (ii), suffer a severe autoinflammatory disease characterised by pustular lesions on the lower ventrum (ii).
- Figure S4. Lymphocytes are not required for thrombocytopenia or autoinflammatory disease in Wdr1rd/rd mice (PDF, 507 KB) -
(A) The rd mutation was crossed onto the Rag2−/− (B and T cell deficient) or Jh−/− (B cell deficient) backgrounds. In both cases, at 3 months of age, mice lacking mature lymphocytes had developed autoinflammatory lesions similar to Wdr1rd/rd counterparts. (B) Peripheral blood platelet counts in 12 week old mice. Thrombocytopenia was observed in the absence of B and T cells. Data represent mean plus or minus SD, with an n of 6 mice of each genotype.
- Figure S5. Splenectomy does not increase platelet counts in Wdr1rd/rd mice (PDF, 200 KB) -
Seven-week-old mice were subjected to splenectomy (Spl) or sham surgery (NT) and platelet counts monitored. Data represent mean plus or minus SD, with an n of 10 mice of each genotype.
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