Fig. 7. In vitro interaction of hARL5 with HP1 and importin-. (A) In vitro interaction of GST-HP1 with hARL5 constructs. hARL5, hARL5(Q80L), hARL5(T35N), hARL5(dC), or hARL5(dMIR) were synthesized by in vitro translation. Samples (5 µl) were mixed with 10 µg of GST or GST-HP1 immobilized on glutathione-Sepharose beads and incubated at 4°C for 1 hour before beads were washed four times with 1 ml of the same buffer. Bound proteins were eluted by boiling in 20 µl of 2x protein sample buffer and separated by SDS-PAGE in 12% gel. Input lane contained 10% of the amount added to beads. Proteins were stained with Coomassie Blue to ensure equal loading, and the bound proteins were visualized by autoradiography. (B) In vitro interaction of GST-importin- with hARL5 constructs. 10 µg of GST or GST-importin- immobilized on glutathione-Sepharose beads were mixed with 5 µl of in vitro translated proteins of interest as described as above.