Easypack tablet

For protein extraction human biopsies were homogenized by peqlab mill (program: 2 × 5000 rpm 20 s with 10 s pause) in lysis buffer (in mM): 50 Tris-HCl, pH 7.5, 120 NaCl, 5 EDTA, 0.1% NP-40, 1 DTT, 1 NaVO₃, 1 NF, 0.2 PMSF, protease inhibitor (cOmplete™ Protease Inhibitor Cocktail, Roche #04693116001) and phosphatase inhibitor (EasyPACK tablet, Roche #4906845001). All steps were performed at 4 °C to avoid native dephosphorylation of the protein extract. The concentration of the supernatant was measured using Bradford Assay (Coomassie Protein Assay Kit, Pierce™ #23200). 45 μg protein of each sample was precipitated for 45 min at 4 °C with one volume 10% TCA. Pellets were washed twice with ice-cold acetone and stored in acetone at −80 °C until use.

For in-vitro dephosphorylation assays, protein was isolated in EDTA-free lysis buffer without addition of phosphatase inhibitor (in mM): 50 Tris-HCl, pH 8, 100 NaCl, 10 MgCl₂, 1% NP-40, 1 DTT, protease inhibitor (cOmplete™ EDTA-free Protease Inhibitor Cocktail, Roche # 4693132001). One half of each sample was treated with phosphatase inhibitor (EasyPACK tablet, Roche #4906845001). The other half was incubated with calf intestine phosphatase (Sigma, #P4978) for 1 h at 37 °C. 45 μg protein of each sample was precipitated for 45 min at 4 °C with one volume 10% TCA. Pellets were washed twice with ice-cold acetone and stored in acetone at −80 °C until use.

For myc tag specific co-immunoprecipitation^{62 (link)} HEK293 cells were lysed in lysis buffer (in mM): 10 Tris-HCl, pH 7.5, 150 NaCl, 0.5 EDTA, 0.5% NP-40, 1 PMSF, protease inhibitor (complete tablet, Roche #04693116001) and phosphatase inhibitor (EasyPACK tablet, Roche #4906845001). 10 µg of total protein extract was used as input on SDS-Page. 20 µL Myc-Trap magnetic beads (ytma-20, Chromotek) were equilibrated in washing buffer (in mM): 10 Tris-HCl, pH 7.5, 150 NaCl, 0.5 EDTA, 0.1% NP-40. 1 mg of protein lysate per sample was incubated with beads over night at 4 °C. Beads were washed and eluted in 30 µL elution buffer (in mM): 200 glycine, pH 2.5. Before SDS-Page followed by immunoblot analysis eluates were neutralized (in mM): 1000 Tris-HCl, pH 8.