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Confirmation of phosphorylation at Thr-34. Wild-type and Thr34Ala MTHFR were expressed in HEK293 cells and purified by immunoprecipitation using antiserum against human MTHFR and protein G-Sepharose. The expressed human MTHFR proteins were detected by Western blotting after SDS/PAGE. Five times as much sample was loaded in B as in A to ensure sufficient sensitivity. Antiserum against human MTHFR and anti-pThr-Pro antibody were used for A and B, respectively.
