Fig. 3. Effect of cAMP on TTF1 phosphopeptides in FRTL5 thyroid cells. FRTL5 cells were deprived of TSH for 3 days in serum-free medium and metabolically labeled with [32P]Pi for 4 h in the presence or absence of forskolin (40 µM), a neutral stimulator of adenylate cyclase. TTF1 was immunoprecipitated from total cell extracts purified by SDS-PAGE and digested with the proteolytic enzyme, thermolysin. The resulting peptides were size-fractionated by two-dimensional TLC, and the label was detected by autoradiography. Arrows, the peptides a and f that are stimulated by forskolin. O, origin. A representative set of three independent experiments that yielded similar results is shown. An identical phosphopeptide map was observed in cells stimulated with either TSH (10 milliunits/ml) or cAMP analogues.