Figure 4. Retinal ganglion cell-5 (RGC-5) cell-line expression of γ-synuclein protein and mRNA were investigated using immunofluorescence and quantitative RT–PCR. A: Immunofluorescent staining of RGC-5 cultures reveals γ-synuclein staining (green) localized in the perinuclear area (arrowheads) and in cell processes (arrows). B: no fluorescence was detected in the absence of primary antibody.Nuclei were counterstained with DAPI (blue). C:qRT–PCR of endogenous synuclein-family transcripts in RGC-5 demonstrates that γ-Synuclein had the highest level of expression (Ct=24.44±0.3), followed by α-synuclein (Ct=26.72±0.3) and finally β-synuclein (Ct=36.93±0.4). Ct represents threshold cycles; a lower number reflects higher levels of initial mRNA. A representative duplicate curves are shown for three independent isolation. Ct differences were statistically significant (p<0.05).