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. 2008 Oct 3;9(12):1222–1229. doi: 10.1038/embor.2008.186

Figure 4.

Figure 4

Chk2 kinase phosphorylation controls Strap stabilization. (A) U2OS or HCT15 cells were transfected with Strap expression vector (500 ng). Cells were treated with vehicle or etoposide (+; 10 μM) for 16 h before collection. Cell extracts were loaded on the basis of β-galactosidase activity and Strap was detected using anti-HA (a). (b) The level of endogenous proteins in the same extracts. Strap underwent a fivefold increase in U2OS cells in (a), and a fourfold increase in (b), which was not apparent in HCT15 cells; n=4. (B) HCT15 cells were transfected with the Strap expression vector (500 ng) in the presence or absence of the Flag-Chk2 expression vector (500 ng). Cells were treated with vehicle or etoposide (+; 10 μM) for 16 h before collection. Cell extracts were loaded on the basis of β-galactosidase. Strap was detected using anti-HA and Chk2 with anti-Flag. Strap underwent 1.8-fold increase without etoposide treatment, and 2.3-fold increase under etoposide treatment on Chk2 expression; n=3. (C) HCT15 cells were transfected with the Flag-Chk2 expression vector (500 ng) as indicated. Endogenous Strap was detected using the Strap antibody and anti-Flag M2 to detect Chk2. PCNA was used as a loading control. Strap underwent 3.7-fold increase without etoposide treatment, and 5.6-fold increase with etoposide treatment, on Chk2 expression when normalized to the PCNA loading control; n=3. (D) U2OS cells transfected with HA-Strap WT (500 ng) were treated with etoposide (+; 10 μM) for 16 h in the presence or absence of Chk2 inhibitor (2 μM). Cell extracts were loaded on the basis of β-galactosidase activity and Strap was detected using anti-HA; n=3. (E) U2OS cells were treated with vehicle control or etoposide (+; 10 μM) for 16 h in the presence (for 2 h) or absence of Chk2 inhibitor (2 μM). Cell extracts were probed for endogenous Strap as described. GAPDH was used as a loading control; n=3. (F) U2OS cells were transfected with the Flag-tagged dominant-negative (D/N), Flag-tagged Chk2 or vector alone (M; 500 ng) as indicated and treated with etoposide (+; 10 μM) for 16 h before collection. Extracts were loaded by total protein content. Strap was detected using the anti-Strap, and dominant-negative Chk2 and Chk2 were detected using anti-Flag. β-Actin was used as a loading control; n=4. Chk2, Checkpoint kinase 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, haemagglutinin; PCNA, proliferating cell nuclear antigen; Strap, stress responsive activator of p300; WT, wild type.