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Asparagine Hydroxylation of the HIF Transactivation Domain: A Hypoxic Switch
David Lando, Daniel J. Peet, Dean A. Whelan, Jeffrey J. Gorman, and Murray L. Whitelaw
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Supplementary Material
Supplemental Figure 1. The wild-type and Gal4DBD/HIF chimeras used in Fig. 3B are expressed at similar levels during transient transfection. Whole-cell extracts of control or transfected cells were separated by SDS-PAGE, and proteins were detected by immunoblotting with polyclonal antibodies directed against the COOH-terminus of HIF-1
(
A) or HIF-2
(
B).
Medium version | Full size version
Supplemental Figure 2. Expression of wild-type and amino acid-substituted HIF-1 proteins during the transient transfection process of Fig. 4. Whole-cell extracts from the indicated untreated or DP-treated transfected cells were separated by SDS-PAGE, and HIF-1 proteins were detected with a polyclonal antibody directed against the HIF-1 COOH-terminus. A similar pattern was obtained for expression of the respective wild-type and amino acid-substituted HIF-2 proteins (data not shown).
Medium version | Full size version
| Supplemental Table 1. Theoretical and observed masses of the indicated ions derived from tandem MS sequencing of the hydroxylated (2106 column) and nonhydroxylated (2090 column) forms of the peptide YDCEVNVPVPGSSTLLQGR.
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| m/z |
| Fragment | Theory | Observed | Difference |
| 2090 | 2090 | 2106 | 2090 | 2106 |
| y1 | 175.119 | 175.118 | 175.118 | 0.001 | 0.001 |
| y2 | 232.140 | 232.142 | 232.140 | 0.002 | 0 |
| y3 | 360.199 | 360.199 | 360.199 | 0 | 0 |
| y4 | 473.283 | 473.279 | 473.278 | 0.004 | 0.005 |
| y5 | 586.367 | 586.272 | 586.363 | 0.005 | 0.004 |
| y6 | 687.415 | 687.410 | 687.402 | 0.005 | 0.013 |
| y7 | 774.447 | 774.447 | 774.440 | 0 | 0.007 |
| y8 | 861.479 | 861.473 | - | 0.006 | - |
| y9 | 918.500 | 918.488 | - | 0.002 | - |
| y10 | 1015.553 | 1015.551 | 1015.550 | 0.002 | 0.003 |
| y11 | 1114.622 | 1114.632 | 1114.622 | 0.01 | 0 |
| y12 | 1211.674 | 1211.678 | 1211.672 | 0.004 | 0.002 |
| y13 | 1310.743 | 1310.730 | 1310.734 | 0.013 | 0.009 |
| y14 | 1424.786 | 1424.769 | 1440.771 | 0.017 | 16.006 |
| y15 | 1523.854 | 1523.834 | 1539.836 | 0.020 | 16.002 |
| y16 | 1652.897 | 1652.919 | 1668.87 | 0.012 | 15.973 |
| y17 | 1812.927 | - | - | - | - |
| y18 | 1927.954 | - | - | - | - |
| y19 | 2091.018 | - | - | - | - |
| | | | | |
| b1 | 164.071 | - | - | - | - |
| b2 | 279.098 | 279.094 | 279.105 | 0.004 | 0.007 |
| b3 | 439.128 | 439.129 | 439.129 | 0.001 | 0.001 |
| b4 | 568.171 | 568.176 | 568.173 | 0.005 | 0.002 |
| b5 | 667.239 | 667.233 | 667.237 | 0.006 | 0.002 |
| b6 | 781.282 | 781.283 | 797.283 | 0.001 | 16.001 |
| b7 | 880.351 | 880.349 | 896.344 | 0.002 | 15.993 |
| b8 | 977.403 | 977.406 | 993.406 | 0.003 | 16.003 |
| b9 | 1076.472 | 1076.467 | 1092.458 | 0.006 | 15.986 |
| b10 | 1173.525 | - | 1189.497 | - | 15.972 |
| b11 | 1230.546 | - | 1246.526 | - | 15.98 |
| b12 | 1317.578 | - | 1333.611 | - | 16.033 |
| b13 | 1404.610 | - | - | - | - |
| b14 | 1505.658 | - | - | - | - |
| b15 | 1618.742 | 1618.69 | 1634.75 | 0.052 | 16.008 |
| b16 | 1731.826 | 1731.832 | 1747.716 | 0.006 | 15.89 |
| b17 | 1859.884 | 1859.89 | 1875.824 | 0.006 | 15.94 |
| b18 | 1916.906 | - | - | - | - |
| b19 | 2073.007 | - | - | - | - |
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