Figure 5.

UbcH7 knockdown increases Chk1 levels and decreases phosphorylated PTEN specifically. Asynchronously growing cells were treated with UbcH7 siRNA or NS siRNA for 72 h and were blotted for proteins indicated. All results were observed in at least two experiments each run in duplicate. (A) Samples from cells depleted of UbcH7 were blotted with α-Chk1, α-Chk2, α-E1, or α-UbcH7 as indicated. The E1 loading control is shown for each immunoblot. (B) Samples were blotted with α-E1, α-E6-AP, α-PTEN, α-UbcH10, or α-UbcH7 as indicated. (C) Left, HeLa cells were treated with siRNA specific for UbcH7 for 72 h and stained for α-Chk1 (top) or α-UbcH7 (bottom). Right, HeLa cells were treated with UbcH7 siRNA or NS siRNA for 72 or 96 h. Percentage of cells containing Chk1 foci were quantified from three separate experiments. *p < 0.05. (D) Samples were blotted with α-P-Ser 280 Chk1, α-P-Ser 380 PTEN (αP-PTEN), α-Akt, α-P-Ser 380/Thr 382/383 PTEN (αP3-PTEN), or α-E1 as indicated. (E) HeLa cells were synchronized at the G1/S boundary by treatment with 2 mM HU for 18 h. Cells were released from drug synchronization, and samples were obtained immediately after treatment (G1), 4 h after release into drug-free media (S phase), or 8 h after release (G2/M) and blotted with α-E1, α-P-Thr 382/383 PTEN (αP3-PTEN) or α-P-Ser 280 Chk1as indicated.