Fig. 2.

E2 and PLD increase phosphorylation of Myc at Ser62 and suppress phosphorylation at Thr58. (A) MCF-7 cells were prepared and collected as described in Figure 1 and placed in serum-free, phenol red-free DMEM with or without E2 (2nM) as indicated. Lysates were prepared and analyzed for the levels of phosphorylated Myc protein at Ser62 by Western blot analysis with an antibody against Myc phosphorylated at this site (Anaspec). The blot was stripped and reprobed with an antibody to total Myc protein. The blot was also reprobed with an antibody to actin to control for loading. (B) MCF-7 cells were treated and collected as in (A) and the lysates were analyzed for the levels of phosphorylated Myc protein at Thr58 using an antibody that recognizes Myc phosphorylated at this site. The blot was stripped and reprobed with antibodies to Myc and actin as in (A). (C) The levels of phosphorylated Myc in presence of E2 were normalized to their respective levels in the untreated control was determined using densitometer quantification. The relative changes in phosphorylated Myc for both Sre62 and Thr58 were compared to level of total Myc protein. (D) MCF-7V and MCF-7P2 cells were prepared and collected as described in (A) and placed in phenol red-free DMEM with 0.5% serum. Lysates were prepared five days later and analyzed by Western blot for the level of Myc phosphorylated at Ser62 (D) and Thr58 (E) as in (A) and (B) respectively. (F) The levels of phosphorylated Myc in the MCF-7P2 cells were normalized to the level in the MCF-7V was determined using densitometer quantification. The relative changes in phosphorylated Myc for both Ser62 and Thr58 were compared to level of total Myc protein. Experiments shown in (A), (B), (D) and (E) are representative of three independent experiments. The data in (C) and (F) are the averages of three independent experiments normalized to the levels of Myc protein. Error bars represent the standard deviation.