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. 2017 Sep 14;10(1):60–73. doi: 10.1093/jmcb/mjx034

Figure 5.

Figure 5

GSK3β phosphorylates E2F1 and facilities E2F1 deubiquitination and stability. (A) A549 cells were transfected with vector or GSK3β mutant with S9A (Gsk3βS9A) for 24 h, and then transfected with HA-tagged E2F1 (HA-E2f1) plasmid for 24 h. Cell lysates were analyzed by IP. (B) A549 cells were treated with CHX (300 μg/ml) with or without TWS119 (10 μM) for 0, 3, 6, and 9 h. Cell lysates were immunoblotted with E2F1 and β-actin antibodies. E2F1 intensities were measured by ImageJ software. *P < 0.01, compared to TWS(−) treated cells. (C) A549 cells were treated with TWS119 (10 μM) for 2 h and analyzed by in vivo ubiquitination assay with a modified IP. (D) A549 cells were transfected with Cont shRNA, Usp11 shRNA, or Gsk3βS9A-HA for 48 h as indicated and analyzed by in vivo ubiquitination assay with a modified IP.