Figure 4

ALK1-induced PP1α negatively regulates transcriptional activity downstream of TGF-β/ALK1.(A) TGF-β kinetics and upregulation of PP1 isoforms on mRNA level. MEECs were stimulated with TGF-β or adenovirally infected with LacZ, caALK1, caALK5 or Smad7 at MOI of 500. Forty hours later, including starvation overnight, the cells were lysed, RNA was isolated and cDNA was prepared. PCR-amplified products for PP1α, β and γ and Id1 are indicated on the right of the figure. β-actin was included as a loading control. (B) MEECs were transfected with siRNA-PP1α using oligofectamine in medium without serum. Twelve hours later the transfection medium was changed to medium with 10% FCS. Forty-eight hours later, the cells were lysed. Whole cell lysate was sonicated, fractionated on SDS-PAGE and subjected to immunoblotting. The filter was incubated with antibodies against PP1α and actin to measure loading of proteins in each sample. (C) Left panel. MEECs were transfected with BRE-luc in the absence or presence of PP1α. Sixteen hours later the cells (where indicated) were transfected with siRNA-PP1α using oligofectamine. Forty-eight hours later, luciferase activity was measured 6 h after stimulation with TGF-β or not. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown. Right panel. MEECs were transfected with (CAGA)₁₂-luc in the absence or presence of PP1α. After 16 h, the cells was transfected with siRNA-PP1α (where indicated) using oligofectamine. Thirty-two hours later, cells were incubated with TGF-β for an additional 16 h, whereafter luciferase activity was measured. Values are corrected for transfection efficiency as measured by β-galactosidase activity. A representative experiment using triplicate samples is shown.