Figure 1.

Formation of an SPL/RGS complex in resting platelets and CHO cells. (A) Western blot analysis of SPL in platelets and brain. Note that all 4 lysates were run on a single gel, and, indicated by the white line, a marker lane and an empty lane were excised. (B) Western blot analyses of RGS10 and RGS18 in lysates prepared from human platelets and human brain. The vertical white line indicates where an intervening lane was excised. (C) Platelet lysates were precipitated with anti-RGS10, anti-RGS18, or rabbit nonimmune immunoglobulin (Ig) and then probed as indicated. (D) Lysates were prepared from CHO cells cotransfected with RGS18 and Myc-tagged SPL (residues 177-817, Myc-SPL). Proteins were precipitated with anti-Myc, anti-RGS18, or nonimmune globulin (Ig), and then probed as indicated. Each example is representative of ≥ 2 experiments. (E) Lysates prepared from duplicate sets of CHO cells transfected with full-length Myc-SPL were incubated with GST (lanes 1 and 2) or GST-RGS18 (lanes 3 and 4). Proteins that became bound to glutathione beads were probed with anti-Myc. (F) Platelet lysates supplemented with GDP ± AlF⁴⁻ as indicated were incubated with GST or GST-RGS18. Proteins that became bound to glutathione beads were probed with anti-G_qα. Experimental details are in supplemental Methods.