Quantification of Protein Modifications in LUHMES Cells

For sample preparation, LUHMES cells were lysed in Laemmli buffer and boiled at 95 °C for 5 min. The lysate was centrifuged for 1 min at 10,000× g through NucleoSpin Filters (Macherey-Nagel, Düren, Germany) to remove nucleic acids. The DigiWest method was used exactly as described earlier [31 (link)]. Briefly, the analysis procedure started with an electrophoretic separation and blotting of proteins onto nitrocellulose membranes. Then, proteins were biotinylated and eluted from small pieces of the membrane (each lane was cut into 96 evenly large strips). Eluted proteins were incubated with color-coded streptavidin-coated Luminex beads (Luminex FlexMAP 3D system). Beads were incubated with primary antibodies for the selected proteins (acetylated tubulin; phosphorylated (at serine-51) and non-phosphorylated eIF2-alpha) and a phycoerythrin-labelled secondary antibody (all reagents and devices by the Thermo Fisher LUMINEX platform (Waltham, MA, USA)).