p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell
Figure 1
Rac1 was isolated as a binding protein of p104. (a) The DNA-encoding p104 (amino acids residue 140–898) was inserted into the EcoRI/Pst Isite of a GAL4 DNA-binding domain vector pGBT9. After colony selection on plates lacking Trp, His, and Leu, β-galactosidase assay was performed on the filter. Rac1 clones were isolated as a binding partner of p104. (b) Using anti-p104 serum, immunoprecipitation was performed with mouse brain extract and analyzed by Western blotting with an anti-Rac1 antibody. Immunoprecipitation using an unrelated antibody (IgG) was used as a negative control. (c) Colocalization of p104 and Rac1 in NIH3T3 cells. NIH3T3 cells were grown on cover slips in 6 well plates and transfected with the pEGFP C1-p104 plasmid. After 24 h, cells were fixed and stained with a Rac1 or lamin antibody followed by a Rhodamine-conjugated anti-rabbit antibody. Fluorescence was observed using a confocal microscope (upper eight panels) or fluorescence microscope (lower eight panels). (d) p104 was specifically associated with Rac1. The carboxy-terminal domain of Rac1, RhoA and Cdc42 was fused with EGFP and transfected into C2C12 cells. After 24 h, cell lysates were immunoprecipitated with anti-p104 serum and Western blot analysis was performed using an anti-GFP antibody. The expression of EGFP-fused Rac1, RhoA, and Cdc42 is shown as a control in the lower panel.
