Journal: Nature communications
Article Title: Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function.
doi: 10.1038/s41467-024-54356-1
Figure Lengend Snippet: Fig. 4 | Compound 4 inhibits eIF4G:eIF4E binding and cap-dependent transla- tion in cell lysate assays. a Lysates from SW620 cells were incubated with 1–100 µM compound 4 or 5 or positive control peptide (RIIY) for 30 min. Endo- genous eIF4E was immunoprecipitated and immunoblotted for eIF4G, 4E-BP1 and eIF4E. Quantitation of 4E-BP1 (b) or eIF4G (c) with endogenous eIF4E in SW620 and HeLa cell lysates, determined by the electro-chemiluminescent binding assay fol- lowing incubation for 30 min with DMSO vehicle (Cont), 100 µM compound 4 or 100 µM RIIY peptide. Complexes were immobilised by an eIF4E antibody and captured eIF4E, eIF4G and 4E-BP1 detected by their respective secondary anti- bodies. Values represent ratios of 4E-BP1:eIF4E or eIF4G:eIF4E electro- chemiluminescence relative to DMSO control (n = 2 biological replicates). d Electro-chemiluminescent assay for binding of eIF4G or 4E-BP1 with eIF4E in SW620 (n = 2 biological replicates), or (e) in HeLa lysates (n = 3 biological replicates, mean ± SD) following incubation for 30 min with compound 4 or 5. Results are expressed as luminescence signals relative to DMSO control. f Quantification of eIF4E:eIF4G interaction in H1299 cells by electro-chemiluminescent assay. Cell lysates treated with RIIY 4E-BP1 derived peptide or RIIG negative control peptide at 0.1–100 µM for 30 min (n = 2 biological replicates). g Quantification of the endo- genous eIF4E:eIF4G interaction in H1299 cell lysates at 0.1–100 µM (for 6 h) of compound 4 or 5, as measured by electro-chemiluminescent assay (mean ± SD from n = 3 biological replicates). h HeLa cell lysates for in vitro translation were incubated for 30 min with 1, 10, 100 µM of compound 4 or 5. Results are expressed as firefly or renilla luminescence signal normalized to DMSO control and expressed as % (mean ± SD from n = 3 biological replicates). Significance was determined using two-sided unpaired t-test comparing compound 4 to compound 5 at each concentration. Statistically significant p-values (p < 0.05) are shown on the plot and source data is located in the Source Data file.
Article Snippet: Monoclonal anti-eIF4E antibody (R&D Systems, Cat#MAB3228, RRID:AB_2097694) was biotinylated using the Lightning-Link kit (R&D Systems Inc. Cat# 371-0010) according to the manufacturer instructions and 20μg used to coat 1ml streptavidinmagnetic beads (Pierce Cat# 88817) for 1 hr at room temperature (RT) with rotatory shaking.
Techniques: Binding Assay, Incubation, Positive Control, Immunoprecipitation, Quantitation Assay, Control, Derivative Assay, Negative Control, In Vitro, Concentration Assay
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