Fig. 5. Interaction of hARL5 with HP1 in the two-hybrid system. (A) Expression of LexA-ARL fusion proteins. Yeast reporter strain L40 was transformed with the indicated LexA construct, pBTM116 (LexA only), or pLexA-lamin. Samples (20 µg) of cell lysates were subjected to SDS-PAGE in a 10% gel, then stained with Coomassie blue (b, upper panel). Proteins were transferred to nitrocellulose and reacted with anti-LexA antibodies (a, upper panel; b, lower panel), or anti-ARL5 antibodies (a, lower panel) as indicated, followed by detection using the ECL system. (B) Interaction of hARL5 and mutants with HP1 in the two-hybrid system. Yeast reporter strain L40 co-transformed with pACT2-HP1 and the indicated pLexA-ARL construct or pLexA-lamin were plated on synthetic histidine-containing medium lacking leucine, tryptophan, uracil and lysine (His⁺ plate, upper panel). Colonies from His+ plates were assayed for ß-galactosidase activity by a filter assay to test for specificity (lower panel). Colonies from His⁺ plates were also patched on His^- selective plates lacking histidine, leucine, tryptophan, uracil, and lysine (His^- plate, middle panel). (C) Interaction of hARL5 and mutants with HP1, HP1ß, and HP1 in the two-hybrid system. Yeast reporter strain L40 co-transformed with pACT2-HP1, -HP1ß, or -HP1, and the indicated pLexA-ARL5 construct or pLexA-lamin were plated and assayed as described above (a). Proteins were transferred to nitrocellulose and reacted with anti-HA antibodies (b, lower panel), followed by detection using the ECL system. Samples (20 µg) of transformed yeast lysates were subjected to SDS-PAGE in a 10% gel, then stained with Coomassie blue (upper panel b). Proteins were transferred to nitrocellulose and reacted with anti-HA antibodies (b, lower panel), followed by detection using the ECL system. Positions of protein standards (kDa) are on the left.