The running buffer was made from 14.4 g/l glycine and pH was adjusted by Tris to 8.5.
Tris buffer was made from Tris base and adjusted to the correct pH with 5 M HCl; MOPS and MES buffers were made by adjusting the pH using 5 M NaOH.
Sodium acetate buffers were made in the pH range from 4.5 to 6.5 with 0.25 pH steps and sodium phosphate buffers were made in the pH range from 5.5 to 8 with 0.5 pH steps.
All buffers were made using Milli-Q water.
RIPA buffer-soluble extracts were made from cells harvested at each time point.
The washed pellets were pooled, dissolved in about 50 mL of phosphate buffer, and washed twice by centrifugation at 100,000 g for 60 min. The final pellet was resuspended in 45 mL of phosphate buffer, and 100 μL aliquots were made from a stirring suspension, which was snap frozen and stored at −70°C.
Tumor lysates were made from orthotopic tumors by mincing the tumor in lysis buffer.
The phosphate buffer for chromatography was made from sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O, 106,346), and disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O, 106,580) was purchased from Merck KGaA (Darmstadt, Germany).
The imaging buffer was made to Nikon protocols from Buffer A (10 mM Tris (pH 8.0 +50 mM NaCl), Buffer B (50 mM Tris-HCl (pH 8.0 +10 mM NaCl+10% Glucose), and GLOX (250 µl: 14 mg Glucose Oxidase+50 µl Catalase (17 mg/ml)+200 µl Buffer A) to make MEA imaging buffer (7.0 µl GLOX, 70 µl 1 M MEA and 620 µl Buffer B).
A stock solution of 1 l of MES buffer (2- N-morpholino) ethanesulphonic acid) was made from 70.4 g 2- N-morpholinohene sulphonic acid (MES free acid) and 193.3 g of MES sodium salt (pH 6.5 – 6.7) and filter sterilized through a 0.2- N-morpholino
