FIG. 1.

Binding of CARM1 to Fli-I in vitro and in vivo. (A) The LRR region (vertical stripes) and two gelsolin-like motifs (black boxes) of Fli-I protein are indicated. (B) In vitro binding assay. [³⁵S]Fli-I protein synthesized in vitro was incubated with GST or GST-CARM1 immobilized on glutathione-agarose beads; bound proteins were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A portion of the in vitro translated Fli-I protein before incubation with the beads is shown at left (Input, 10%). The position of the 145-kDa full-length Fli-I protein is indicated. (C) Coimmunoprecipitation assay. pSG5.Flag-FliI(LRR) or pSG5.Flag-FliI(gelsolin) (2.5 μg) was transiently transfected along with pSG5.HA-CARM1 or pSG5.HA-PRMT1 (2.5 μg) into Cos-7 cells. Immunoprecipitation (IP) was performed on transfected-cell extracts with antibodies against the Flag epitope, and the precipitated proteins were analyzed by immunoblotting (W) with anti-HA antibodies (upper panel). A sample of the transfected-cell extract before immunoprecipitation (2% of the volume used for the immunoprecipitation shown in lane 3) is shown in lane 1. The relative expression of HA-CARM1 and HA-PRMT1 before immunoprecipitation is shown in the lower panel. Results are representative of two independent experiments. LRR, LRR fragment of Fli-I; GEL, gelsolin-like fragment of Fli-I; and IgG, position of the immunoglobulin heavy chain on the immunoblot.