Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 Page 5 of 10 Figure 3 DYRK1A is required for efficient phosphorylation of 12E8 tau. Shown in A are results of siRNA screening for DYRK1A siRNA. 12E8 tau is shown in red and total tau is shown in green (see Experimental Methods). For B, H4 cells overexpressing four repeat tau (4R0N) were transfected with siRNA targeting the DYRK1A transcript. Silencing of DYRK1A was confirmed with anti-DYRK1A antibody (top panel). Percent control values represent the average of three independent siRNA transfections and westerns. In A and B, NS refers to the nonsilencing control. identify those kinases that significantly affected both 12E8 and total tau levels simultaneously. To identify those candidates, we pulled out kinases that had statisti- cally significant effects on both total tau levels and 12E8 tau levels relative to controls, irrespective of the result- ing effects on the ratio of 12E8 tau/total tau. These are presented in Table 2. Interestingly, two siRNAs to the eukaryotic translation initiation factor 2 a kinase 2 (EIF2AK2) gene significantly reduced 12E8 and total tau levels (Figure 5 and Table 2). These siRNAs reduced 12E8 tau levels to 61 ± 8% (p = 0.012) and 72 ± 7% (p = 0.018) of non-silencing siRNA controls and reduced total tau levels to 65 ± 10% (p = 0.026) and 71 ± 8% (p = 0.025) of control values, respectively. The comparable eductions of both phosphorylated and total tau levels led to no change in the ratio of 12E8 tau/total tau. To confirm these screening results for EIF2AK2, we retransfected our cell line with EIF2AK2 siRNA. Wes- tern blotting results confirmed that EIF2AK2 is required for maintaining normal levels of 12E8 tau and total tau Figure 4 AKAP13 is required for efficient phosphorylation of 12E8 tau. Shown in A are results of siRNA screening for AKAP13 siRNA. 12E8 tau is shown in red and total tau is shown in green (see Experimental Methods). For B, H4 cells overexpressing four repeat tau (4R0N) were transfected with siRNA targeting the AKAP13 transcript. Silencing of AKAP13 was confirmed with anti-AKAP13 antibody (top panel). Percent control values represent the average of three independent siRNA transfections and westerns shown. In A and B, NS refers to the non-silencing control.