eIF4E was digested out of HaloTag-eIF4E using SgfI and PmeI, and then ligated into pFN33K (Promega) to obtain the LgBiT-eIF4E construct. 4E-BP1 was cloned into pFN35K (Promega) using the same method to obtain the SmBiT-4E-BP1 construct. HaloTag-4E-BP1 and -eIF4E have been described elsewhere. 27 (link),44 (link),45 pFN33K LgBiT-eIF4E and pFN35K SmBiT-4EBP1 (50 ng each) were reverse-transfected into MCF-7 and MDA-MB-468 cells in a 96-well white opaque plate using Lipfectamine LTX with PLUS reagent. After 16 h, cells were arrested with nocodazole (500 nM). 20 h later, cells were treated with rapamycin (100 nM) and/or palbociclib (5 μM). After 2 h incubation, Nano-Glo Live cell reagent (Promega #N2011; 25 μL) was added and total luminescence was read within 40 min on a BioTek Cytation 3 reader.
Protocol detail
Bioluminescent Assay for eIF4E-4E-BP1 Interaction
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Corresponding Organization : University of Michigan–Ann Arbor
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Variable analysis
independent variables
- Rapamycin (100 nM)
- Palbociclib (5 μM)
- Total luminescence
- MCF-7 and MDA-MB-468 cell lines
- Nocodazole (500 nM) for cell cycle arrest
- Nano-Glo Live cell reagent (Promega #N2011) for luminescence detection
- BioTek Cytation 3 reader for luminescence measurement
- None specified
- None specified
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