Figure 5
Smad7 recruits PP1α to ALK1. (A) COS-7 cells were transfected with eGFP-PP1α, wtALK1/HA and/or Flag-Smad7. After 48 h at 37°C, cells were lysed. Whole cell lysates were immunoprecipitatedwith HA antibody, fractionated by SDS-PAGE, and subjected to immunoblotting with PP1c antiserum. Total lysates were fractionated by SDS-PAGE and subjected to immunoblotting with antisera against Flag, PP1 and HA to check expression levels. (B) Left panel. Phosphatase inhibition assay. MEECs were adenovirally infected with the indicated constructs and immunopreciptated with HA antibody. Kinase assay was performed with 2.5 μCi of [32P]γ ATP per sample at 30°C for 30 min. Samples were washed in lysis buffer before they were incubated in a phosphatase buffer with or without the PP1 inhibitor, I-2 at 37°C for 60 min. The samples were separated by SDS-PAGE and phosphorylation quantified using phosphoimager. Right panel. Fraction of the total lysate was immunoblotted with HA antiserum to check expression levels of the receptor.