Figure 2.

NM23 as the source of Rad-GAP activity and its association with Rad in vivo. (A) Human skeletal muscle extracts were prepared as described in Experimental Procedures and were subjected to two rounds of immunoprecipitation (IP) with an anti-human nm23 antibody. The immunocomplex and the cytosol before and after nm23-depletion were used for the GAP assay as described in Fig. 1. (B) C2C12 myotubules overexpressing Rad were solubilized and subjected to immunoprecipitation for 2 hr with anti-Rad (1:100) and anti-nm23 (1:50) antibodies, then were subjected to SDS/PAGE and immunoblotting with both antibodies. Starting from the left, lanes 1 and 2 represent an IP with anti-Rad antibody; lanes 3 and 4 with anti-nm23 antibody; and lanes 5 and 6 represent straight blotting.