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. 2010 Feb 24;285(19):14671–14680. doi: 10.1074/jbc.M109.066118

FIGURE 2.

FIGURE 2.

Knockdown of p35 decreases CDK5 activity in SAOS-2 Cells. A, efficacy of lentivirus-based shRNAs for p35 was tested by cotransfecting pCMV-p35 and pLKO-p35shRNA-17, -18, -20, or control empty vector into SAOS-2 cells. Cell lysates were collected 2 days after transfection and were immunoblotted for p35. B, pSVE or pSVE-Rb was cotransfected with pLKO-shp35–18 or pLKO vector into SAOS-2 cells. Cells were selected with puromycin (0.5 μg/ml) 2 days after transfection, cell lysates were collected on day 5 and immunoprecipitated with anti-Cdk5 antibody to perform an in vitro kinase assay using histone H1 as the substrate. Cell lysates were also analyzed for CDK5 and pRB expression by immunoblotting. Numbers at the bottom of panels show ratio of kinase activity in each lysate to control lysate.