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Figure 2


Fig. 2. mDia1 co-immunoprecipitates with POPX2. (A) Protein lysates from COS7 cells were incubated with Protein G Sepharose beads covalently coupled with random IgG or affinity-purified anti-POPX2 antibodies. The eluates were analyzed by SDS-PAGE and western blotting using anti-mDia1 antibodies. Lane 1, COS7 total-protein lysate; 2, eluate from co-IP with random IgG; 3, eluate from co-IP with anti-POPX2 antibodies. (B) Dot-blot using 10 µg of MBP as control protein, 1 µg 6xHis-tagged mDia-DN and 10 µg 6xHis-tagged mDia-DN. The nitrocellulose membrane was dotted with MBP and 6xHis-tagged mDia-DN and incubated with bacterial-produced GST-POPX2. Far-western analysis was done using anti-GST antibodies to detect direct binding. (C) COS7 cells transfected with mDia-DN or mDia-{Delta}C were co-transfected with POPX2 or RhoV14 to determine the binding by co-IP and western blotting. Lane 1, HA-POPX2 + Flag-mDia-DN; 2, HA-POPX2 + Flag-mDia-{Delta}C; 3, HA-RhoV14 + Flag-mDia-DN; 4, HA-RhoV14 + Flag-mDia-{Delta}C.