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Blood, Vol. 107, Issue 5, 1892-1895, March 1, 2006
Anti-Epo receptor antibodies do not predict Epo receptor expression
Blood Elliott et al.
107: 1892
Supplemental materials for: Elliott et al
Files in this Data Supplement:
- Figure S1. Effects of shRNA knock-down of EPOR transcripts on expression of proteins detected by M-20 and C-20 antibodies (JPG, 34.2 KB)
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SH-SY5Y cells were infected with a retrovirus expressing shRNA that targets the human EPOR sequence AAATGGTTGCTGCCCCGGAAC. Protein extracts from cells expressing the shRNA, or from control cells, were prepared and subjected to Western blotting. (A) Transfection of SH-SY5Y cells with shRNA reduced the expression of the 59-kDa protein detected by M-20. (B) There was no effect of the EPOR shRNA on expression of the 66- and 100-kDa proteins detected by C-20. (C) Cyclophilin B was used as a loading control. The positions of the molecular weight markers are illustrated on the left (kDa).
- Figure S2. Lack of specificity of anti-EpoR antibodies (JPG, 70.8 KB)
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Cell extracts were prepared from 3 × 106 UT-7/Epo cells and immunoprecipitated with 2 µg C-20 or rabbit IgG. Immuno-complexes were washed three times with cell lysis buffer, and all of the material was loaded onto the gel and subjected to Western immunoblotting. Lane 2 contained extracts from 50,000 UT-7/Epo cells. Blots were probed with C-20, 07-311, and M-20 antibodies. Cell extracts from COS-7 cells expressing FLAG-EpoR were used as a positive control for EpoR (Lane 1). Compared to UT-7/Epo cell lysates, the C-20/protein immunocomplexes were enriched in the 37-, 59-, and 100-kDa proteins while the 66-kDa protein was greatly reduced in intensity (A). The M-20 and 07-311 antibodies detected the 59-kDa protein but not the 37-, 66-, or 100-kDa proteins in the C-20 immunocomplexes (B,C). Lane 1, FLAG-EpoR; Lane 2, UT-7/Epo cell extract; Lane 3, cell extract immunoprecipitated with C-20; Lane 4, cell extract immunoprecipitated with rabbit IgG. The arrows indicate the positions of the 66-kDa protein, FLAG-EpoR/EpoR and IgG heavy chain. The positions of the molecular weight markers are illustrated on the left (kDa).
- Figure S3. Expression of the 59-kDa protein in EPOR(+/+) but not EPOR(-/-) mice (JPG, 38.9 KB)
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Extracts were prepared from cells and tissues and subjected to Western immunoblotting using M-20. Extracts from COS-7 cells expressing FLAG-EpoR and from 32D/Epo cells that express functional EpoR (but without the FLAG sequence) were used as positive controls for EpoR. The arrow indicates the position of the 59-kDa protein. The positions of the molecular weight markers are illustrated on the left (kDa). To obtain EPOR-knockout [EPOR (-/-)] mouse embryos, timed matings were performed with heterozygous EPOR (+/-) mice25: EPOR(+/+) and EPOR(-/-) embryos were recovered at day 12.5 for histology of the complete embryo and at day 13.5 for liver tissue. M-20 detected a protein similar in size to FLAG-EpoR in 32D-Epo cells and in fetal liver cells from wild-type, EPOR(+/+), mice (lanes 3,4). The protein was absent in fetal liver cells from EPOR(-/-) mice (lane 5). The presence of the 28-kDa protein in all tissues suggested that this protein was not derived from the EPOR gene. All experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Amgen.
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