Fig. 9. In vivo interaction of hARL5 with HP1. For immunoprecipitation of the HP1-hARL5 complex, COS-7 cells were co-transfected with an hARL5 construct and HP1. After 48 hours, cells were harvested, washed three times in PBS, suspended in PBS, and placed on ice. DSP was then added. After 1 hour at 0°C, the reaction was stopped. The cells were harvested, washed and lysed. After centrifugation (10,000 g, 15 minutes) of lysates, M2 anti-Flag affinity gel was added to the supernatants, followed by incubation at 4°C for 3 hours with agitation. Beads were then washed three times with TBS and dispersed in appropriate amounts of sample buffer. Eluted proteins were subjected to western blotting analysis using anti-GFP polyclonal antibody (lower panel). Samples of total cell lysates were also subjected to western blotting analysis using anti-Flag and anti-ARL5N antibodies (upper two panels).