Skip to main content
. 2013 Jul 29;288(37):26678–26687. doi: 10.1074/jbc.M113.466433

FIGURE 3.

FIGURE 3.

NDR1/2 and cyclin D1/Cdk4 cannot phosphorylate each other in vitro. A, 293T cells were transfected with pCMV-Myc-Cdk4 or pCMV-Myc-Cdk4 D158N. The cell lysates were immunoprecipitated with anti-Cdk4 antibody. The beads were incubated with 1 μg of GST-Rb(773–832), GST-NDR1 K118A, or GST-NDR2 K119A in the presence of [γ-32P]ATP and then assessed by SDS-PAGE, followed by autoradiography of the gel. The asterisk indicates the expected position of GST-NDR1 K118A and GST-NDR2 K119A. B, 1 μg of GST-p21, GST-cyclin D1, or GST-Cdk4 was incubated with GST-NDR2-PIFtide in the presence of [γ-32P]ATP for in vitro kinase assay. The samples were assessed by SDS-PAGE, followed by autoradiography. The asterisk indicates the expected position of GST-cyclin D1 and GST-Cdk4. C, 293T cells were transfected with pCMV-Myc-cyclin D1/Cdk4 alone or with pCMV-FLAG-NDR1/2. The cell lysates were immunoprecipitated with anti-Cdk4 antibody. The beads were incubated with 1 μg of GST-Rb(773–832) in the presence of [γ-32P]ATP for in vitro kinase assay. aa, amino acids; CBB, Coomassie Brilliant Blue.