Figure 1. Over-expression of ARF6-DNM attenuates agonist-internalization of both the P2Y₁ and P2Y₁₂ purinoceptor.

1321N1 human astrocytoma cells stably expressing either HA-tagged P2Y₁ and P2Y₁₂ purinoceptor were transiently transfected with either constitutively inactive mutant forms of ARF6 (ARF6 T27N; ARF6-DNM) or ARF1 (ARF1 T31N; ARF1-DNM). Over-expression of DNM forms of ARF1 or ARF6 was confirmed by Western Blotting which showed a 4–5 fold increase over endogenous levels of ARF proteins (data not shown). Cells were subsequently treated with ADP (10 µM; 0–30 min) and surface receptor loss assessed by ELISA (Fig. 1A and Fig. 1B) or receptor internalization visualized by confocal immunofluorescent microscopy (Fig. 1C). In Figure 1A and 1B the data represent means ± SEM of five independent experiments. *p<0.05 compared with respective vector alone controls (Mann–Whitney U-test). In Figure 1C receptor cell surface expression was assessed by examining co-localization with a plasma membrane marker (CellMask™ Plasma Membrane Stain).