alphascreen surefire kit  (Revvity)

Journal: Nature Communications

Article Title: Engineering an autonomous VH domain to modulate intracellular pathways and to interrogate the eIF4F complex

doi: 10.1038/s41467-022-32463-1

Figure Lengend Snippet: a Western blot analysis of 293FT cells transfected for 48 h with FLAG tagged empty vector (Mock), 4EBP1 and anti-eIF4e VH mutants, in whole cell lysate (left, WCL) and FLAG immunoprecipitation analysis (right). b The NanoBit assay consists of a split luciferase fused either to eIF4E or eIF4G 606–646 , whose interaction restores luciferase function , . Exogenous expression of antagonists of the eIF4E:4G interaction result in a decrease in total luminescence. SmBiT-eIF4E and eIF4G 604–646 -LgBiT constructs were co-transfected into HEK293 cells with either empty vector (Mock) or vectors containing 4EBP1 or VH mutants (as indicated) for 48 hrs. After 4 h of incubation, the luciferase activities of the reconstituted NanoBit protein were measured in living cells as described in Material and Methods. All values represent mean ± standard deviation ( n = 3, independent experiments). c A bicistronic luciferase reporter (left panel), which measures the relative amount of cap-dependent translation (Renilla) to cap-independent translation (Firefly), was co-transfected with indicated plasmid. Renilla and Firefly luciferase activity was measured 48 h post transfection and plotted as a ratio-metric value. The Renilla luciferase is controlled by a GC rich 5’UTR whose activity correlates to cap dependent translation, whilst the Firefly luciferease is regulated by an internal ribosomal site (IRES). Lysates were prepared from the transfected cells and blotted for the presence of the respective constructs (below left-hand graph). In the right-hand graph, the lysates from the bicistronic assay were probed for eIF4E serine 209 phosphorylation eIF4E with the Alpha Surefire assay (Perkin-Elmer). This assay is a no wash sandwich bead-based immunoassay that enables quantification of eIF4E phosphorylation levels. All values represent mean ± standard deviation ( n = 3, independent experiments). d Hela cells were transfected with indicated DNA using different amounts (µg) for 48 h and levels of indicated protein measured with western blot. Protein loading was assessed with β-actin antibody. E) Lysates from the transfected HELA cells in d were also used to perform m 7 GTP pull down followed by western blot analysis. Levels of 4E-BP1 4 ala and VH domain mutants were detected using anti-FLAG. All western blot analyses were repeated twice to verify their reproducibility. Representative blots are shown. Source data for a , b , c , d and e are provided in the Source Data file. Center line and limits denoted on dot blots represent the mean value and standard deviation of the replicates, respectively.

Article Snippet: In the right-hand graph, the lysates from the bicistronic assay were probed for eIF4E serine 209 phosphorylation eIF4E with the Alpha Surefire assay (Perkin-Elmer).

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Luciferase, Expressing, Construct, Incubation, Standard Deviation, Activity Assay, Phospho-proteomics, Bead-based Assay