Fig. 3. Deletion mutants of Runx2 express at comparable levels and exhibit unaltered DNA binding activity. (A) Runx2 deletion mutants. The black box shows highly conserved runt homology domain (RHD). The dark gray box represents the amino acid sequences in Runx2 homologous to the NMTS of Runx1 whereas the C-terminal light gray box represents the highly conserved VWRPY domain. (B) HeLa cells grown in 100 mm plates were transfected at 50-60% confluence with 10 µg of the expression construct for each of the mentioned deletion mutants. The cells were harvested 24 hours after transfection in direct lysis buffer and proteins were separated by SDS-PAGE. The western blotting was carried out as described. The blots were incubated with monoclonal antibody against HA tag (Santa Cruz; dilution 1:3000) followed by incubation with HRP-conjugated secondary antibody raised against mouse (Santa Cruz; dilution 1:2000) to detect the proteins. Cdk2 antibody (1:5000) was used as an internal control. (C) HeLa cells transiently expressing mentioned deletion mutants were processed for nuclear extract preparation. Radioactively labeled Runx consensus sequence was used as probe. The mentioned proteins were used in increasing concentrations, that is, 2, 4 and 8 µg in electrophoretic mobility shift assay; EV (empty vector) indicates that cells were transfected with vector backbone.