Effect of PK activation on the cleavage of HK. HUVECs were preincubated with 20 nmol/L ¹²⁵I-HK for 1 hour at 37°C. Unbound ¹²⁵I-HK was removed and either buffer (A) or 20 nmol/L PK (B) was added for 1 to 30 minutes. The cells were washed and solubilized in electrophoresis sample buffer containing 2% β-mercaptoethanol. The proteins were boiled, separated on 10% SDS-PAGE, and analyzed by autoradiography. The numbers to the left of the photographs represent molecular mass standards in kilodaltons. The numbers at the bottom of the gels refer to the time (in minutes) that the cells were incubated with PK. CN refers to ¹²⁵I-HK directly added to the gel.