Fig. 6: PP4 is active toward pSer666 and pThr806 and negatively regulated by Cdk9.

a PP4R2 was immunoprecipitated from HCT116 cell extracts, incubated in vitro with purified, recombinant Cdk9/cyclin T1 and ATP, and immunoblotted with antibodies to PP4R2-pThr173, total PP4R2, or PP4C, as indicated at right. b HCT116 cells were mock treated (DMSO) or treated with inhibitors of Cdk9 (NVP-2) or Cdk1 (RO-3306), as indicated, for 1 h before lysis and extract preparation. Anti-PP4R2, anti-PP4C, or control IgG immunoprecipitates, as indicated, were incubated with Spt5-derived phosphopeptides containing pSer666 or pThr806, as indicated, and phosphate release was measured colorimetrically. Error bars indicate ±s.d. of six biological replicates (n = 6). c Anti-PP4R2 immunoprecipitates were incubated with 5 ng purified, recombinant Cdk9/cyclin T1 and ATP or mock treated (as indicated), washed and tested for phosphatase activity toward an Spt5-derived phosphopeptide containing pSer666 or pThr806, as in b. Activity of Cdk9-treated samples is plotted as a fraction of activity of the corresponding, mock-treated immunoprecipitate (defined as 1.0). Error bars indicate ±s.d. from three biological replicates (n = 3). Individual data points from biological replicates are shown in the plots (b and c). d HCT116 cells were infected with lentivirus expressing shRNA targeting PP4C (three different hairpins, sh1-3) or a nontargeted control vector (V), and chromatin fractions were immunoblotted for Spt5-pSer666, Spt5-pThr806, total Spt5 (to ensure equal loading),and total PP4C (to assess efficiency of depletion). Source data are provided as a Source data file.