4.4. Analysis of Binding to the Bromodomains of BRG1 and BRM

The BRD-binding activity of the small molecule PFI-3 to the BRD of the catalytic subunits of SWI/SNF was assessed by an adaption of a previously described CETSA procedure [22]. MT330 and LN229 GBM cells were transduced with a lentivirus encoding either an epitope-tagged BRG1 or BRM BRD. GBM cells expressing the BRG1 BRD construct were treated with PFI-3 (30 mM) or DMSO as vehicle control for 2 h. After heating over a temperature range from 44.5 to 55.6 °C for 5 min, the cells were lysed, placed on ice, and then immunoblotted for BRG1 or actin. As a complementary approach, we next developed a chromatin-based assay for BRD binding [17]. A GFP-tagged BRG1 BRD containing a nuclear localization signal was expressed in MT330 GBM cells by lentiviral transduction. After transduction, stable pools of cells were treated PFI-3 (10 or 30 μM), or DMSO as a vehicle control. After 2 h, the cells were fixed, DAPI counterstained, and examined by confocal microscopy (Zeiss model LSM700).