GRK2 and GRK6 expression in 1321N1 cells and platelets. (A) 1321N1 cells were transfected with 5 μg DNA containing empty vector or DNM-GRK constructs. Whole cell lysates from these cells or human platelets were subjected to SDS-PAGE, followed by immunoblotting with GRK-specific primary, as detailed in “Materials and methods.” Data shown are representative of 3 experiments. (B) Effect of siRNA treatment on endogenous GRK expression in 1321N1 cells. 1321N1 cells stably expressing P2Y₁₂ purinergic receptors were transfected with scrambled, GRK2-, or GRK6-specific siRNAs twice in a 24-hour interval, and the cells were harvested after 4 days. GRK2 expression was analyzed by immunoblotting using an anti-GRK2 monoclonal antibody, whereas GRK6 expression was analyzed using an anti-GRK6 polyclonal antibody. These results are representative of at least 3 similar experiments. Similar results were obtained in 1321N1 cells stably expressing P2Y₁ purinergic receptors (data not shown).