Expression and localization of the wild-type and mutant hTTP in transfected human cells.

<p>(A) Immunoblotting. HEK293 cells were transfected with pHis-hTTP plasmids. Proteins in the soluble extracts (10,000<i>g</i>, 10 µg/lane) were separated by SDS-PAGE (10% Tris-glycine gel) and transferred onto nitrocellulose membrane. The membrane was incubated in anti-MBP-hTTP serum (1∶10,000 dilution, 2 h) followed by secondary antibodies (1∶10,000 dilution, 1 h). The blot was incubated in Super Signal for 5 min and exposed to X-ray film for 5 sec. The underlined numbers in the plasmids 1–9 below the gel represent the sites of serine/threonine residues mutated to alanine residues in addition to the mutations of hTTP in the preceding plasmid. (B) Immunostaining. HEK293 cells were transfected with pBS+ control plasmid and pHis-hTTP plasmids encoding wild-type His-hTTP and mutant His-hTTP with S(214,218,228)A and S(88, 90, 93, 197, 214, 218, 228, 296)A mutations. The cells were stained with anti-MBP-hTTP antibodies (1∶5,000 dilution, overnight) and labeled with goat anti-rabbit Alexa Fluor 488 (1∶1,000 dilution, 1 h). Immunofluorescence was recorded by confocal microscopy.