Ph strips

The standard compounds L-glutamine, D-glutamine, L-glutamic acid, HPLC grade methanol and acetonitrile, 18-crown-6, acetone, trifluoroacetic acid, and concentrated sulfuric acid were purchased from Sigma-Aldrich corporation, USA. Anhydrous DMF, acetonitrile extra dry and cesium bicarbonate, were purchased from Acros Organics, USA. All the gases hydrogen (HY R300), helium (HE R300) and anhydrous ammonia (AM AH200N705) were purchased from Airgas Inc. USA. Deionized Ultra-Filtered Water (DIUF), ethyl ether anhydrous stabilized with BHT were purchased from Fisher scientific, USA. Ethanol 200 proof was purchased from Decon laboratories Inc. PA, USA. All the chemicals were used without any further purification. The seppak cartridges, C18 plus, tC18 vac 3 cc, tC18 plus short and Silica plus were purchased from Waters corporation, USA. The precursor (S)-tert-butyl-2-((tert-butoxycarbonyl)amino)-4-iodobutanoate was purchased from ABX, Advanced biochemical compounds, Germany. Millex GS 0.22 μm filters were from Merck Millipore Ltd. pH strips were from EMD Millipore Corporation, MA, USA. Semi-preparative HPLC column (Macherey-Nagel, VP250/10 Nucleodur C18, HTech, 5 μm was obtained with Synthra HCN Plus synthesis module which was purchased from Synthra GmbH, Hamburg, Germany. QC HPLC column Astec CHIROBIOTIC™ T chiral HPLC column was purchased from Sigma-Aldrich Corporation, USA.

Sequential extraction of tau protein was performed to test the effect of MARK4 on tau protein solubility. We performed fractionation analysis as previously described for PS19 mice.^{34 (link),37 (link)} Briefly, one brain hemisphere was homogenized in 5 vol high-salt reassembly buffer (HS-RAB: 100 mM MES, pH 7.0, 1 mM EGTA, 0.5 mM MgSO₄, 0.75 M NaCl and 0.1 mM EDTA + proteinase inhibitors and phosphatase inhibitors) using a Teflon pestle homogenizer, and the homogenate was centrifuged at 50 000 × g in an Optima MAX-TL ultracentrifuge (Beckman Coulter, Brea, CA, USA) for 40 min at 4°C to collect the supernatant as an HS-RAB soluble fraction. The pellet was homogenized in 1 M sucrose/RAB buffer, and the solution was centrifuged at 50 000 × g for 20 min, followed by pellet homogenization in 1 vol RIPA buffer and centrifugation at 50 000 × g for 20 min at 4°C to collect the supernatant as a RIPA soluble fraction. Next, we extracted a RIPA-insoluble pellet with 1 vol of cold 70% formic acid solution and centrifuged it at 15 800 × g for 20 min at 4°C to collect the supernatant as an FA soluble fraction. The FA fraction was diluted in 1:10 (v/v) neutralization buffer (1 M Tris base and 0.5 M Na₂HPO₄), and the pH was checked using pH strips (Merck). All fractions were processed for western blotting as described above.