Transfection of Cdk5/p25 into HEK293 cells identifies NMHC-B as a substrate for Cdk5. A. Western blot showing pSerCdk substrate, Cdk5 and p25 levels in cells transfected with Cdk5/p25, Cdk5/p25 transfected cells treated with 1 μM Roscovitine, cells transfected only with p25 or Cdk5 and untransfected cells. A 200 kDa band appeared only in the Cdk5/p25 double transfected cells. This band decreased in intensity when treated with 1 μM Roscovitine and did not show up in single transfected or untransfected cells. B. Western blot showing pSerCdk substrate, Cdk5 and p25 levels in cells transfected with 2, 1.5 or 1 μg of each plasmid and in cells treated only with transfection reagent, Lipofectamine. The cells transfected with 1.5 μg of Cdk5 and p25 plasmid, i.e.3 μg total plasmid DNA, showed sufficient phosphorylation of Cdk substrates and least effect on cell morphology and viability (data not shown). C. Western blot showing a pSerCdk antibody immunoprecipitated sample from Cdk5/p25 transfected HEK293 cells probed with pSerCdk substrate antibody and with antibody to NMHC-B. Detection of the same band by both antibodies verifies that the 200 kDa band is NMHC-B.