Figure 3. Inhibition of ARF6 activity in 1321N1 cells attenuates internalization of both the P2Y₁ and P2Y₁₂ purinoceptor.

1321N1 human astrocytoma cells stably expressing either HA-tagged P2Y₁ and P2Y₁₂ purinoceptor were incubated with either (A) penetratin-coupled-myristoylated (Myr) inhibitory ARF peptides (myr-ARF1-peptide and myr ARF6-peptide) or penetratin vehicle alone (Vehicle) or (B and C) SecinH3 (15 µM) or vehicle control for 30 minutes prior to study. In (A) and (B) cells were subsequently treated with ADP (10 µM; 30 min) and surface receptor loss assessed by ELISA. The data represent means ± SEM of five independent experiments. *p<0.05 compared with respective control (Mann–Whitney U-test). In (C) inhibition of P2Y-stimulated ARF6-GTP levels by SecinH3 treatment was assessed. Cells expressing either HA-tagged P2Y₁ and P2Y₁₂ purinoceptor were pretreated with SecinH3 (15 µM) or vehicle control for 30 minutes prior to study. Total ARF6 expression in the cell lysates and ARF6-GTP precipitated with GST-GGA3 VHS-GAT beads were detected by immunoblotting using an anti-ARF6 mouse monoclonal. The relative intensity of each ARF6-GTP band was normalised to total ARF6 and measured by densitometry. Values are mean ± SEM from three separate experiments. *p<0.05 compared with no ADP treatment alone control (Mann–Whitney U-test).