Generation and characterization of a monoclonal anti-ING4 antibody.

<p>(<b>A</b>) Schematic diagram of ING4 protein containing a nuclear localization signal (NLS) and a plant homeodomain (PHD). Stick figures represent the ING4 fragments that were used to immunize mice. (<b>B</b>) N-terminal (AA 5–147) and C-terminal (AA 173–249) recombinant fragments of ING4 purified from bacteria visualized by SDS-PAGE gel stained with Coomassie Blue. (<b>C</b>) Detection of ING4 and HA-epitope tagged ING4 overexpressed in 293T cells by Western blot using BTIM-4 and anti-HA monoclonal antibody. * denotes smaller ING4-derived protein species recognized by the antibody. (<b>D</b>) Western blot analysis of ING4 protein expression using BTIM-4 antibody in MCF10A breast epithelial cells and T47D breast cancer cells containing pMIG (the vector control), ING4 (ING4 overexpression), shNT (non-targeting shRNA control), or shING4 (shRNA targeting ING4). Tubulin antibody was used as a loading control. (<b>E</b>) MCF10A cells transduced with pMIG (the vector control), ING4 (ING4 overexpression), shNT (non-targeting shRNA control), or shING4 (shRNA targeting ING4), and T47D cells transduced with pMIG (the vector control) or ING4 (ING4 overexpression) were immunostained with BTIM-4 anti-ING4 antibody (red) and visualized using fluorescent microscopy. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain individual cell nuclei (blue). White scale bars represent 100 µm.