FIG. 2.

Interaction of p204 with Id1, Id2, and Id3. (A) Comparison of the levels of the Id1, Id2, and Id3 proteins in murine thigh muscle. Id1, Id2, and Id3 were immunoprecipitated from a thigh muscle extract of 15-day-old C129 mice by using polyclonal antibodies to the Id1, Id2, or Id3 proteins immobilized on protein A-agarose beads. The proteins were eluted from the washed beads, and the eluate was subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and blocked with nonfat milk. The Id1, Id2, and Id3 proteins were detected by using antibodies to Id1, Id2, and Id3, respectively, and the ECL system. (B) Binding of p204 to GST-Id2 determined by pull-down assay in vitro. [³⁵S]methionine-labeled p204 translated in a reticulocyte lysate (IVT204) (lane 1) bound to GST-Id2 (lane 4) but not to GST (lane 2) or GST-FHF1B (lane 3) immobilized on glutathione-Sepharose beads. The amount of p204 used in the experiment in lane 1 was 1/10 of that in the experiments in lanes 2 and 3. The bound proteins were analyzed by gel electrophoresis and fluorography. The position of the p204 band is indicated. The GST-FHF1B protein was used as a negative control. (C) Left panel, purified GFP (lane 1) or GFP-204 fusion protein (lane 2) was subjected to SDS-10% PAGE, electrotransferred to a nitrocellulose membrane, denatured, and renatured. After blocking with bovine serum albumin, the membrane was incubated with purified GST-Id2 protein, followed by incubation with anti-Id2 antibodies and visualization. Right panel, the same membrane was stripped and reprobed with anti-GFP antibodies. The GST-Id2 band (as retained by GFP-204) and the IgG band are indicated by arrows. (D) Interaction of p204 with Id2 in vivo, assayed by coimmunoprecipitation with anti-p204 antiserum. Extracts prepared from C2C12 myoblasts in GM or in DM for 1 day (DM1) or 2 days (DM2) were immunoprecipitated (IP) with anti-p204 antiserum (lanes 2 to 4). The immunoprecipitates and the cell extract (lane 1) were examined by immunoblotting with anti-Id2 antibodies. The amount of lysate used in the experiment in lane 1 was 10% of that used in the experiments in lanes 2 to 4. The Id2 and IgG bands are indicated. (E) Binding of p204 to His-Id1 and His-Id3 determined by pull-down assay in vitro. GST-204 was incubated with HisTrap beads alone (lane1) or with HisTrap beads loaded with His-SAP1 (lane 2) His-Id1 (lane 3), or His-Id3 (lane 4). The beads were washed and eluted, and the proteins released were analyzed by immunoblotting with anti-p204 antibodies. The GST-204 band is indicated. (F) Interaction of p204 with Id1, Id2, and Id3 in vivo, assayed by coimmunoprecipitation with antisera to Id1, Id2, and Id3. Extracts prepared from C2C12 myoblasts in DM for 1 day were immunoprecipitated with control IgG (lane 3) or antiserum to Id2 (lane 2), Id1 (lane 4), or Id3 (lane 5). The immunoprecipitates, together with cell lysate (lane 1) (20% of the amount used for immunoprecipitation), were examined by immunoblotting with an anti-p204 antiserum. The p204 and IgG bands are indicated. For further details, see Materials and Methods.