Figure 2 of Faralli, Mol Vis 2019; 25:237-254.

Figure 2. Expression of the RAC1-specific GAP CHN1 is upregulated by DEX. A: Schematic diagram showing the two splice variants of α-chimaerin. Both variants contain a GAP domain that is specific for RAC1 and a C1 domain that mediates binding to diacylycerol and phorbol esters. The α2-chimaerin also contains a SRC homology 2 domain (SH2). PCR showed that both HTM and TM-1 cells express mRNA for the α2-chimaerin variant. In contrast, brain tissue expressed mRNA for the α1-chimaerin variant. Primers used to distinguish between the variants were previously described [57]. The forward and reverse primers, respectively, used to detect the α1-chimaerin variant were 5′- AAA ATG CCA TCC AAA GAG TCT-3′ and 5′- GAA ATT GTG AAT CTT TTC ATA TTT-3′. The forward and reverse primers, respectively, used to detect the α2-chimaerin variant were 5′-GGC TCT ACT ACG ATG GCA AGC-3′ and 5′- CTG TAG AAT CTC TCT CAT CAT GT-3′. B: qPCR analyses showed that DEX caused the upregulation of CHN1 mRNA compared to the EtOH-treated and no trt controls. The increase at day 2 was statistically significant (*) at p<0.05 compared to the no trt controls. Data are presented as the mean ± SEM. The mRNA levels were normalized to the no trt group. 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 an increase in α-chimaerin levels was detected in the DEX treated cells. 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 the phagocytosis of pHrodo™ bioparticles (red) in TM-1 cells overexpressing α2-chimaerin was similar to mock transfected cells. Cells were counterstained with CellMask Green to visualize the cells. E: Quantification of the number of cells that phagocytosed pHrodo™ bioparticles in cultures transfected with FLAG tagged-α2-chimaerin (n=4216 cells) did not show a statistically significant change (p<0.8) in phagocytosis compared to mock transfected cells (n=1279 cells). Data are presented as the percent positive ± SEM.