Figure 2
Src promotes estrogen-dependent ERα degradation.
(A) ERα before and 6 hours after addition of estradiol (Est) with or without the proteasome inhibitor n-acetyl-Leu-Leu-norleucinal (LLnL) to estrogen-depleted MCF-7 cells. Equal loading was confirmed by β-actin. (B) ERα t1/2 was assayed by CHX chase in estrogen-depleted cells and at 2, 4, and 6 hours after addition of estradiol. Graph shows results of densitometric analysis of 3 CHX chase experiments (mean ± SEM). (C) Cells were grown in 0.1% cFBS for 48 hours and then treated with estradiol alone, 5% cFBS plus estradiol, or 5% cFBS alone. ERα and β-actin were assayed 6 hours later. (D) Serum- and estrogen-deprived MCF-7 cells were transferred to 5% FBS plus estradiol with or without added Src inhibitor PP1, and ERα was assayed 6 hours later. (E and F) MCF-7 cells were transfected with PCI-Src Y530F (Src) or empty vector (Mock). After 24 hours, (E) ERα and Src levels were assayed and (F) ERα t1/2 was assayed by CHX chase (mean ± SEM). (G and H) The MCFpINDSrc2 line was estrogen depleted for 72 hours, and Src was induced or not for 24 hours with PA prior to addition of estradiol. (G) Src at time of estradiol addition (left), and ERα and β-actin before and 6 hours after estradiol (Est + or –) was added (right). (H) CHX pulse chase, starting 6 hours after estradiol addition with (+Src) or without (–Src) prior induction of Src by PA.
