Figure 5. SFRS7 acts as a tau exon 10 splicing enhancer in the glial cell-line U- 373 MG.

(A) Knockdown of endogenous SFRS7 protein was determined with quantitative RT-PCR showing a mean 9,13 fold (mean ΔΔCt 3.19) decreased endogenous mRNA expression for SFRS7. Bars are mean ± SEM.* indicate P<0.05 (one sample t-test with theoretical mean 0). N = 4, each sample was measured in triplicate. (B) Knockdown of endogenous SFRS7 protein was determined with a SFRS7 specific antibody by western blot using total cell protein extracts obtained from SFRS7 siRNA and scrambled transfected U-373 MG cells. An antibody specific for actin was used as a loading control. Westernblot image is a representative of at least 3 independent experiments. (C) Ratio numbers were obtained by dividing endogenous 4R and 3R tau mRNA concentrations from U373-MG cells transfected with siRNA SFRS7 or scrambled siRNA. Endogenous 4R and 3R mRNA concentrations were obtained as described in the material and methods section. A significant decrease of the 4R/3R tau ratio was seen for siRNA SFRS7 compare to scrambled siRNA transfected cells. P-values were calculated using two-tailed unpaired t-test, error bars represent ± SEM, * indicate P<0.05. N = 4, each sample was measured in triplicate. With the use of densitometry on obtained westernblot images (D) a similar significant decrease of the 4R/3R tau ratio was seen on a protein level (D, right graph). Significance was calculated by combining the densitometry data from 3 independent experiments. P-values were calculated using two-tailed unpaired t-test, error bars represent ± SEM, * indicate P<0.05