Figure 7. 32P-labelling of the WT and mutant TTP proteins in intact cells.
WT and nine mutant TTP proteins were labelled with [32P]orthophosphate in HEK-293 cells (A–C). The labelled proteins were either purified with Ni-NTA affinity beads and eluted with imidazole buffer (A, C) or immunoprecipitated with anti-MBP-hTTP antibodies (B). A parallel experiment without 32P was performed, and 10000 g supernatants from the HEK-293 cells were used for immunoblotting with anti-MBP-hTTP antibodies (D). The protein identities are shown at the top of each gel lane. Lane ‘0’ on each side of the immunoblot (D) contained the non-fusion TTP purified from E. coli. (A) and (B) are autoradiographs and panels (C) and (D) are immunoblots. The underlined numbers above the gel lanes represent the sites of serine/threonine residues mutated to alanine in addition to the mutations of TTP in the preceding lane. Note: S197A/S228A is shown as S(197,228)A (etc.).