For tau analysis, extracts of the hippocampus were incubated with alkaline phosphatase mix (500 mM Tris HCl pH 9.0, 500 mM MgCl2, 0.1 M DTT (Invitrogen), 10,000U/ml calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA)) at 37 °C overnight for dephosphorylation.
In order to avoid the participation of primers and unincorporated dNTPs in the subsequent minisequencing primer extension reaction, 2.5 μl of mPCRA and 2.5 μl mPCRB products were combined and treated with an Exo I/Antarctic Phosphatase mix consisting of 5 units of E. coli exonuclease I (Exo I, New England Biolabs) and 1 unit of Antarctic phosphatase (New England Biolabs).
All PCR products (5 µl) were aliquotted onto 384-well microtiter plates and were treated with 2 µl of Shrimp Alkaline Phosphatase (SAP) mix for 20 minutes at 37°C to dephosphorylate unincorporated dNTPs.
Protein concentration of cell lysates was measured by Bradford assay and equal amounts of protein (10 15 µg) were incubated in a phosphatase reaction mix containing 50 mM HEPES (pH 7.5), 0.1% β-ME and 10 mM pNPP (Fluka) for several minutes at 37°C.
For protein phosphorylation analysis, a phosphatase inhibitor mix (GE Healthcare) was also added.
For pooling-deconvolution assays, kinase pools were prepared in Buffer System I. The phosphatase reaction mix contained 400 Unit/ml of lambda protein phosphatase (New England Biolabs), 50 mM Tris-HCl pH7.5, 0.1 Na2EDTA, 5 mM DTT and 0.01% Brij35.
Amplicons were subsequently treated with an exonuclease I/shrimp alkaline phosphatase (SAP) mix to remove PCR primers and dephosphorylate dNTPs, followed by heat inactivation and direct use of CpG-specific primer extension (see workflow Figure 1).
To analyse the ATG16L1/clathrin heavy-chain interaction, HeLa cells were lysed on ice for 30 min in Buffer B (10 m m Tris HCl, 150 m m NaCl, 1 m m EDTA, pH: 8.0), supplemented with protease and phosphatase inhibitors mix, in the presence of 1% Triton X-100.
EBs were isolated in RIPA cell lysis buffer containing a cocktail of protease inhibitors (Protease Inhibitors and Phosphatase Inhibitors mixes used as recommended, Calbiochem, LaJolla, CA) at different time points.
Restriction enzymes, phosphatase, DNA polymerase mix, and T4 DNA Ligase were obtained from New England Biolabs (Ipswich, MA).
Neurons (200,000 cells/well) were lysed in RIPA buffer containing proteinase and phosphatase inhibitor cocktail mix (Sigma-Aldrich) and insoluble debris was removed by centrifugation.
