Fig. 4. A, identification of TTF1 phosphorylated peptides. HeLa cells were transiently transfected with expression vectors coding for wild-type TTF1 or various serine/threonine -> alanine mutants (20) . Transfected cells were metabolically labeled for 4 h with [32P]Pi. TTF1 was immunoprecipitated with specific polyclonal antibodies, purified by PAGE, and digested with thermolysin. wt, wild-type TTF1. B, diagram illustrating the location of specific mutations and phosphopeptides. The mutants have alanine substitutions at the following sites: 1, serine 107; 2, serines 4, 12, 18, and 23 (loss of peptide b); 3, serine 255 (loss of peptides e and partly d); 4, serines 4, 12, 18, 23, 107, 255, and 328 (loss of peptides b, d, and e); 5, serines 328 and 337 (loss of peptides a, d, and f); 6, serines 4, 12, 18, 23, 107, 255, and 337 (loss of peptides b, c, d, e, and f); 7, serines 4, 12, 18, 23, 328, and 337 (loss of peptides a, b, c, d, and f). Peptide f is a fragment of peptide a. Mutant 8, serines 4, 12, 18, 23, 255, 328, and 337, is not phosphorylated in vivo (data not shown).