Figure 3. Expression of the dual GEF/GAP ABR is unaffected by DEX, but it plays a role in phagocytosis. A: Schematic diagram showing that ABR contains both a GEF and a GAP domain. It addition, it contains a pleckstrin homology (PH) domain and a C1 domain that mediates binding to diacylycerol and phorbol esters. B: qPCR analyses showed that 5 d of DEX treatment caused the downregulation of mRNA for ABR compared to the no trt group. The decrease at day 5 was statistically significant (*, p<0.05) compared to the no trt group but not compared to EtOH-treated cells at day 5. The mRNA levels were normalized to the no trt group. Data are presented as the mean ± SEM. All five HTM cell strains (N27TM-2, N27TM-4, N27TM-5, N27TM-6, and N25TM-8) were used for the qPCR analyses; n=5. C: Western blot analyses showed that protein levels of ABR were unaffected by DEX compared to EtOH-treated controls. β-actin was used as a loading control. Equal amounts of protein from the DEX- and EtOH-treated cell lysates were loaded. The figure is a representative blot done on cell lysates from three separate experiments using the HTM cell strain (N25TM-8). n=3. D: Fluorescence micrographs showed a marked decrease in the uptake of pHrodo™ bioparticles (red) in TM-1 cells transfected with ABR siRNA compared to NT siRNA transfected cells. Cells were counterstained with CellMask Green to visualize the cells. E: Quantification of the number of TM-1 cells that took up pHrodo™ bioparticles. Cultures transfected with siRNA to ABR showed a statistically significant (*p<0.05) reduction in phagocytosis (n=3580 cells counted) compared to cells transfected with NT siRNA (n=1460 cells counted). Data are presented as the percent positive ± SEM.