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Blood, Vol. 111, Issue 6, 3229-3235, March 15, 2008
Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins
Blood Takeda et al.
111: 3229
Supplemental materials for: Takeda et al
Files in this Data Supplement:
- Figure S1 (JPG, 42.1 KB)
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(A) Anti-PHD2 Western blots for bone marrow, spleen, liver and kidney of Phd2 CKO and Phd2f/f control mice. (B) Survival rates of Phd2 CKO or Phd2f/f control mice after Phd2 gene disruption by tamoxifen administration.
- Figure S2. Identification of PHD1 and PHD3 protein bands (JPG, 40.9 KB)
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(A) Identification of PHD1. In vitro transcription/translation (IVTT) products were used for autoradiography (top) or anti-PHD1 Western blotting (bottom). Labeling on top of the gel is explained below: “-,” negative control of IVTT reaction (no Phd1 cDNA input); “Phd1 wild,” IVTT reaction where a pBluescript plasmid containing wild-type Phd1 cDNA insert was used; “Phd1  Exon3,” Phd1 cDNA lacking exon 3-encoded sequence. As can be seen in the top panel (35S labeled protein bands), IVTT of wild type Phd1 cDNA generated two bands, where were absent in the “-” lane, indicating that both bands are PHD1 isoforms. As expected, deletion of the exon 3-encoded region generated two faster migrating bands. The identity of the third weak band is not clear, but may be a degradation product. In the bottom panel, we demonstrate that anti-PHD1 reacted with three bands: the top two bands correspond to the two IVTT bands revealed in autoradiography whereas the fastest moving band appeared to be non-specific. Deletion of exon 3 sequence reduced the molecular mass of PHD1 bands, as expected. (B) Identification of PHD3. Experimental design and gel labeling are similar to (A), expect that Phd3 cDNA was used.
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