Fig. 7. PKC phosphorylation and time course of recruitment into DIGs. (A) SaI/A^k and SaI/A^k tr cells were stimulated or not via I-A^k for 15 minutes, lysed and fractionated on a sucrose gradient. DIG fractions were migrated on SDS-PAGE, transferred on PVDF and immunoblotted with the specific phospho-PKC Ab. (B) Kinetics of I-A^k recruitment, PKC- and phospho-PKC recruitment to DIGs were analyzed in SaI/A^k cells. SaI/A^k cells were stimulated via I-A^k for the indicated times, lysed and fractionated on a sucrose gradient. DIG and soluble fractions were immunoblotted with the corresponding Abs for PKC- and phospho-PKC, and western blots were analyzed by densitometric quantification. Time point 0 is arbitrarily set to 1 in both experiments. The time course of I-A^k recruitment to DIGs was determined by SDS-PAGE under boiled conditions followed by 10.2.16 mAb immunoblotting.