Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 Page 3 of 10 Figure 1 Quantitative immunofluorescence detection of reductions in 12E8 tau and total tau protein levels using MAPT siRNA. Shown in A is reduction of both pS262 tau and total tau levels using an siRNA directed at the tau transcript (all splice variants). Using this siRNA, pS262 tau and total tau levels are reduced to 45% and 54% of control values, respectively, following 4 days of transfection with siRNA. pS262 tau is visualized in red. Total tau is in green. Blue staining represents DAPI stained nuclei. Note that significant total tau expression remains following siRNA treatment, likely resulting from the extremely high constitutive levels of tau expression in this cell line. Shown in B is secondary quantitation of the MAPT siRNA effects on 12E8 and total tau using western analyses. Results support the immunofluorescence quantitation shown in A. Representative images are shown. Percent control numbers (± SD) in A represent the average of three independent assays. Westerns in B were performed in triplicate. NS refers to the non-silencing siRNA control. Figure 2 Quantitative reduction of 12E8 tau with siRNA against MARK2. Shown in A is detection of pS262 tau and total tau levels following transfection with MARK2 siRNA. Our quantitative assay detects significant reduction of pS262 tau using siRNA directed at the microtubule affinity regulating kinase 2 (MARK2) gene (red panels; p = 0.029 for three replicates). MARK2 siRNA has no effect on the overall levels of total tau protein (green panels), demonstrating that this assay can specifically detect reductions in pS262 tau. Representative images are shown. Percent control numbers represent the average of three independent assays. NS refers to the non-silencing siRNA control. In B, qRT-PCR results confirmed >95% knockdown of MARK2 mRNA relative to non-silencing siRNA control (NS).