Figure 1.

The N-terminus of coilin mediates self-interaction. Coilin self-interacts in the yeast two-hybrid system (A), as well as by coimmunoprecipitation in vitro (B) and from cell extracts (C). In panel A, various coilin baits, indicated on the left, were cotransformed into the yeast strain PJ69–2A with coilin prey. Interaction was defined as the ability of PJ69–2A containing both bait and prey to grow on selective medium within 3 days. The (+) sign denotes robust growth after 3 days incubation, whereas the ({minus}) sign represents no growth. The lack of coilin prey interaction with aa 92–161 or the C-terminal 214 aa of coilin verified the specificity of the interaction. The interactions were quantified by performing liquid β-galactosidase assays, as described in MATERIALS AND METHODS. Values are reported in Miller units and varied by no more than 20%. (B) Coilin and EE-coilin were in vitro translated with or without [³⁵S]methionine, respectively. Cold EE-coilin (5 μl) was mixed with 5 μl ³⁵S-labeled coilin and immunoprecipitated with anti-EE antibodies (lane 2). Lane 3 contains 1/15th the amount of ³⁵S-labeled coilin used in the reactions. No ³⁵S-labeled coilin was recovered when immunoprecipitated in the absence of EE-coilin (lane 1) or in the presence of an EE-tagged heterologous sequence (HS, lane 4). (C) Cell extracts from GFP-coilin transfected HeLa cells were subjected to immunoprecipitation with anti-GFP antibodies, followed by SDS-PAGE and western blotting with antibodies against coilin. Normal rabbit serum was used as a negative control for immunoprecipitation. Lane 3 contains 10 μl of total cell lysate (TCL) from GFP-coilin transfected cells. IgG(H) is the immunoglobulin heavy chain. (D) Cell extracts from myc-coilin(94–576) transfected HeLa cells were immunoprecipitated with antimyc antibodies, followed by SDS-PAGE and western blotting with antibodies against coilin. No endogenous coilin is observed in lane 2, corresponding to the IP using antimyc antibodies. Lane 1 contains 10 μl of total cell lysate (TCL) from myc-coilin(94–576) transfected cells. Normal mouse serum was used as a negative control for immunoprecipitation (our unpublished results).