Azorsa et al. BMC Genomics 2010, 11:25 http://www.biomedcentral.com/1471-2164/11/25 overexpresses four repeat tau protein, a non-physiologi- cal condition. However, if in subsequent experiments these results extrapolate to mammalian brain, it would suggest that a therapeutic strategy to decrease tau hyperphosphorylation in AD and other tauopathies may require the simultaneous modulation of several phos- phorylation sites. This is one reason why candidate tar- gets that reduce total tau levels, such as EIF2AK2, are appealing since they are likely to simultaneously affect multiple phosphorylation sites. Untangling the complex regulatory networks of tau hyperphosphorylation is a daunting task. However, our results provide an impor- tant first step in defining the scope of kinases and asso- ciated proteins that may be involved in phosphorylating tau on a site highly relevant to tau dysfunction, tau pathology, and AD. Further work remains to determine the complex pathways through which the kinases identi- fied here may interact to control tau phosphorylation levels. Methods Generation of 4R0N tau overexpressing H4 cell line The Human H4 neuroglioma cell line (ATCC) was transfected with a 4R0N tau construct in pcDNA3.1. Following positive selection using geneticin (Invitrogen), individual clones were isolated and screened for tau expression using immunocytochemistry with a rabbit anti-tau antibody (Dako). A single stable tau overexpres- sing cell line was selected for subsequent siRNA studies. Cells were maintained in Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin, geneticin (0.25 mg/ml), and 2 mM L-Glutamine (Invi- trogen). Cells were split 1:10 at 90% confluency, twice a week. High Throughput siRNA Kinome Screen Preparation of siRNA library plates Chemically synthesized siRNA (18.6 μg) from Qiagen’s validated human kinase siRNA Set 2.0 were printed into 96-well black clear bottom plates (Corning #3904) using a BioMek FX (Beckman Coulter). Printed plates were then foil sealed and stored at -80°C until use. High Content siRNA Screening Assay In order to identify genes involved in modulation of tau phosphorylation, we developed a high content siRNA screening assay. Briefly, 4R0N tau overexpressing H4 cells were reverse-transfected with library siRNA and siLentfect (Bio-Rad) using semi-automated transfection as follows: Screening plates were thawed and siRNA was complexed by addition of 50 μl of diluted siLentfect in OptiMEM (Invitrogen) using a μFill (BioTek) followed by incubation for thirty minutes at RT. Cells were tryp- sinized and resuspended in growth media without peni- cillin-streptomycin and added at a concentration of Page 8 of 10 3000 cell/well in 50 μl using a μFill followed by incuba- tion for 5 minutes at room temperature (RT). Assay plates were incubated at 37°C and 5% CO2, for 96 hours. Plates were washed twice with 1× Tris-buffered saline (TBS) (Fisher) and fixed for 15 minutes with4% parafor- maldehyde (PFA) at RT. Plates were then washed twice with 1× TBS and incubated with blocking buffer (TBS with 5% normal goad serum, 0.2% sodium azide, 1% bovine serum albumin (BSA) and 0.1% NP-40) for 1 hour at RT. Fixed cells were next incubated with 1 μg/ml of phospho-tau antibody 12E8 (Elan Pharmaceuticals) at 4°C overnight. Plates were washed three times with 1× TBS-T (TBS with 0.1% Tween-20) followed by incubation with 1:200 dilution of rabbit anti-human tau (Dako) for 1 hr at RT. Plates were then washed twice with 1× TBS-T fol- lowed by incubation with a secondary antibody cocktail of 2 μg/ml FITC-Goat anti-rabbit IgG, 2 μg/ml Cy5- Goat anti-mouse IgG (Jackson Immunore search) and 10 μg/ml Hoechst 33342 (Invitrogen) in blocking buffer for 30 min- utes at RT. Plates were then washed once with TBS-T fol- lowed by twice with TBS leaving the last wash in the plates. Plates were stored at 4°C overnight prior to analy- sis. Plates were imaged and then analyzed on the INCell 3000 (GE Healthcare). The analysis module (Object Inten- sity 01) identified each cell by the blue staining of the nuclei, and measured both red (Cy5) and green (FITC) intensities in a 4 pixel ring around the nuclei. Western Blotting 4R0N tau overexpressing H4 cells were reverse-trans- fected with siLentfect complexed with target siRNA in 6 well plates. Cells were grown for 72 hours at 37°C, 5% CO2. Cell lysates were prepared using the Complete Lysis-M, EDTA-free kit (Roche Applied Science) and quantitated using the BCA protein assay (Pierce). Protein from lysates (20 μg) were separated by SDS-PAGE and transferred to nitrocellulose. Membrane was blocked in 5% BSA for one hour at RT. Membranes were probed with primary antibody overnight at 4°C on a rocker. Membranes were subsequently washed with TBS-T and probed with secondary antibody 1:25000 dilution of HRP-GAM (Jackson Immunoresearch) for forty-five min- utes. Membranes were further washed and developed with Super Signal West Femto Maximum Sensitivity Sub- strate Kit (Promega) and imaged. To test multiple pri- mary antibodies, membranes were stripped for 15 minutes at RT using ReBlot Plus Mild Antibody Stripping Solution (Millipore). Membranes were then washed again for 5 minutes at RT then blocked for one hour in 5% BSA. Membrane was reprobed overnight at 4°C with an anti-Tubulin antibody (1:25000; ICN Biomedicals, Inc.). Antibodies used for detection included anti-tau (1:1000; Dako) primary antibody, 12E8 antibody (1:7500), anti- EIF2AK2 (1:1000; Abcam), anti-DYRK1A (1:500; Abcam), and anti-AKAP13 (1:2000; Bethyl Laboratories, Inc).