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Supplemental figures

Supplemental figures. Tang et al. A. Anti-flag. B. C. D. Tang_ Fig S1. Figure S1. ATM is required for IR-induced PP1 activation.

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Supplemental figures

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  1. Supplemental figures Tang et al

  2. A Anti-flag B C D Tang_ Fig S1

  3. Figure S1. ATM is required for IR-induced PP1 activation. Previously, it was reported that IR activates PP1 in an ATM-dependent manner. The conclusion was drawn by immunoprecipitating endogenous PP1 to observe an increase in the phosphatase activity after IR. However, it is possible that immunoprecipitation of endogenous PP1 might have brought down other phosphatases (associated with PP1) that can be activated by IR. To role out this possibility, we constructed a mammalian expression vector that expresses flag-tagged wild-type or phosphatase-inactive (Aspartic Acid 95 mutated to Alanine-D95A) form of PP1, in an attempt to use D95A as a negative control for immunoprecipitation and phosphatase assays. We transfected the constructs into HeLa cells and assessed the expression of the flag-tagged proteins. While D95A mutant PP1 has significantly diminished phosphatase activity in the absence of DNA damage and IR does not increase it, wild-type PP1 has at least a two-fold increase in phosphatase activity after IR (S1A). Using the phosphatase inactive form of PP1 as a negative control, we confirmed that PP1 is indeed activated after exposure to IR. We also found that only the nuclear fraction, but not the cytoplasmic portion of PP1, was activated in response to IR-induced DNA damage (S1B). An ATM-dependent PP1 activation was observed in human fibroblast lines either deficient (GM9607) or proficient (GM0637) in ATM expression and cells expressing a dominant negative form of ATM (S1C and D). Taken together, these findings support the conclusion that functional ATM is required for activation of PP1 after IR.

  4. H2O S43(ng) H2O S43p(ng) 50 100 50 100 Ser43p Ser43 Figure S2. The specificity of the anti-phospho-Serine 43 antibody. A Western blot of binding of the antibody to peptides that are phosphorylated or unphosphorylated at Serine 43. Tang_ Fig S2

  5. Figure S3: ATM phosphorylation of I-2 is required for Histone H1 dephosphorylation. HeLa cells were transfected with vector, or Xpress-tagged wild-type or S43A mutant I-2. 36 hours after transfection, cells were mock-treated or irradiated and subjected to flow cytometry analyses using an anti-phospho-Histone H1 antibody and propidium iodide. Error bars represent +/-1 SD, graphed are the mean of three independent experiments. Tang_ Fig S3

  6. Figure S4. IR-induced PP1 activation is correlated with inhibition of H3 phosphorylation. PP1 activity and Histone H3 Serine 10 phosphorylation were assessed at indicated time points in HeLa cells treated with 0 or 6Gy of IR. Tang_ Fig S4

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