Staufen1 rescues GFP-CUG200 protein expression. (A) Schematic representation of the GFP-CUG5 and GFP-CUG200 transgenes. (B) C2C12 cells were transfected with GFP-CUG5 or GFP-CUG200 reporter plasmids alone or with the Staufen1 (Stau1) construct. Cells were fixed and analyzed by fluorescent microscopy. GFP, green; DAPI, blue. Bar, 50 µm. (C and D) GFP levels of individual cells were measured by FACS analysis. Representative FACS measurements of GFP fluorescence (GFP-positive [+ve] cells in R3 box). Data are means ± SEM, n = 3. (E, top) Global GFP protein levels from transfected cells were also analyzed by Western blotting using anti-GFP antibodies. Tubulin was used as a loading control. (bottom) Quantification of GFP from Western blotting. Data are means ± SEM, n = 4. (F) Global GFP RNA levels were analyzed by qRT-PCR quantification using primers specific for GFP. Cyclophilin B was used to normalize the data. No difference in mRNA levels was detected between GFP-CUG200 with and without the presence of Staufen1. Data are means ± SEM, n = 4. **, P < 0.01; ***, P < 0.001; ns, P > 0.05 (Student’s t test).