Lapatinib, An Unequivocable Flexibility!

Phosphorylated STAR protein was undetectable in untreated Y1 and Kin-8 cells (Figure 1A) and 8Br-cAMP or ACTH treatment resulted in the appearance of pSTAR protein in only in the Y1 cells. The pSTAR/tSTAR ratio was 4-fold greater after8Br-cAMP treatment compared to ACTH S6 Kinase treatment (Figure 1B). These data demonstrate for the first time that newly synthesized STAR is not phosphorylated in Kin-8 cells. Figure 1 PhosphoSTAR protein expression in Y1 cells. Y1 and Kin-8 cells were placed in serum-free medium overnight then treated for 2 and 6 h in serum-free medium in the absence (?) or presence of either 1 mM 8Br-cAMP or 150 nM adrenocorticotropic hormone ... To assess whether cAMP-PKA signaling contributes to STAR protein stability, we compared the half-life of STAR between the Y1 and Kin-8 cell lines. Using a standard pulse-chase radiolabeling approach, the cells were treated with 8Br-cAMP for 2 h in the presence of [35S]-methionine followed by a cold chase for 4 h in the absence of 8Br-cAMP. STAR protein was recovered by immunoprecipitation and radiolabeled STAR detected by fluorography (Figure 2A). STAR protein half-life in Y1 and Kin-8 cells was determined to be 1.5 �� 0.11 h and 2.2 �� 0.06 see more h, respectively (p LY294002 �� 0.13 h in the presence of ACTH compared to 1.4 �� 0.05 h in the absence of ACTH. Together the data indicate that the degradation of STAR 30 kDa protein in Y1 cells is not regulated by a PKA-dependent mechanism. Figure 3 cAMP-PKA activation and mitochondrial STAR protein half-life in Y1 cells. Y1 cells were radiolabeled with [35S]-methionine for 6 h in the presence of 150 mM ACTH followed by cold chase in either the absence or presence of ACTH for the indicated times. ... 3.2. Precursor STAR Expression in Y1 Cells Y1 cells were treated with 8Br-cAMP either in the absence or presence of the 26S proteasome inhibitors epoxomicin (Epox) or MG132 and phosphoSTAR (pSTAR) and total STAR (tSTAR) protein were detected by Western blot analysis of isolated mitochondria. Addition of either MG132 or Epox resulted in accumulation of precursor STAR in the 8Br-cAMP-treated cells (Figure 4A, tSTAR panel).