FIGURE 5. Rac1_DN Increases Surface Expression of Kir2.1 Measured with TIRF Microscopy.

Cells were transfected with the cDNA for a N-terminal CFP-tagged Kir2.1 in the absence or presence of Rac1_DN. A) Representative images from cells transfected with CFP cDNA viewed with epifluorescence (left: Epi) or TIRF (right) microscopy. All images have been scaled to the same intensity. B-C) Representative images from cells transfected with CFP-Kir2.1 cDNA and either a control plasmid (B) or Rac1_DN (C) cDNA viewed with TIRF microscopy. Images have been scaled to the same intensity. Inset shows scale bar used to calibrate intensity. D) Quantitative analysis of CFP-Kir2.1 intensity under TIRF. Images were background-subtracted and analyzed using ImageJ software. Asterisks denote significance determined by non-parametric ANOVA and Dunn's post-hoc test (*, p < 0.01 and **, p < 0.001). E) Representative immnoblot for total HA-Kir2.1 protein in untransfected cells (UT) or cells transfected with HA-Kir2.1 cDNA and either pcDNA3 (Ctrl) or Rac1_DN cDNA. Left: Staining with anti-HA antibody. Right: Blots were stripped and reprobed with antibody against actin. F) Bar graph shows mean optical density (OD) measurements made using ImageJ. OD measurements for anti-HA bands were normalized to OD measurements for anti-actin bands to control for protein loading and expression levels (n=3). Asterisk indicates significance (p > 0.05) using unpaired Student's t-test.