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

Figure 3.

Figure 3

Strap phosphorylation in cells. (A) Strap was immunoprecipitated (IP) from U2OS cells using anti-Strap or the nonspecific (NS) control. Strap immunoprecipitates were immunoblotted (IB) using anti-phosphoserine and then reprobed for Strap using the anti-Strap; the asterisk indicates a nonspecific band; n=2. (B) U2OS cells were treated with etoposide (10 μM) and Chk2 inhibitor (15 μM) as indicated. Strap was immunoprecipitated as described above, and immunoblotted with anti-phosphoserine. Input Strap levels are shown; the asterisk indicates a nonspecific band; n=2. (C) Flag-Strap was induced in cells using doxycyline. Cells were treated with etoposide (10 μM) and Chk2 inhibitor (15 μM) as indicated. Strap was immunoprecipitated using Flag-tagged beads and immunoblotted with the anti-phosphoserine. The blot was then reprobed with Flag antibody for Strap levels. Input levels represent 5% of the total used in immunoprecipitation; n=2. (D) U2OS cells were transfected with expression vectors (1 μg) encoding wild-type Strap, S221A or empty vector and treated with etoposide (10 μM for 16 h) before collection. Cell extracts were immunoprecipitated as described above with anti-HA and immunoblotted using anti-phosphoserine and then reprobed for ectopic Strap using anti-HA. Strap was phosphorylated fivefold over S221A; n=2. Chk2, Checkpoint kinase 2; HA, haemagglutinin; Strap, stress responsive activator of p300; WT, wild type.