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. 2021 Oct 11;12:5919. doi: 10.1038/s41467-021-26222-x

Fig. 2. FBXL2 interacts with the kinase domain of EGFR and its membrane-targeting function is essential for the destabilization of EGFR protein.

Fig. 2

a A schematic representation of FBXL2 deletion mutants used in this study. b HEK293T cells were co-transfected with Flag-EGFR and an indicated HA-FBXL2 expressing plasmids. After grown overnight, cells were treated with MG132 for 4 h before harvesting for IP-Western analyses. c A schematic representation of EGFR deletion mutants constructed in this study. d HEK293T cells were co-transfected with FBXL2 and an indicated Flag-EGFR expressing plasmids. After grown overnight, cells were treated with MG132 for 4 h before harvesting for IP-Western analyses. e The binding affinity of the recombinant FBXL2/SKP1 protein and EGFR kinase portion was measured by Biolayer interferometry (BLI) assay. The KD values were shown. f HEK293T cells were co-transfected with HA-FBXL2 and indicated Flag-EGFR expressing plasmids. Cells were grown overnight and treated with MG132 for 4 h, followed by IP-Western analyses. g, h HEK293T cells were co-transfected with wild-type Flag-EGFR or Flag-EGFRE931A in the presence of HA-FBXL2 or a vector control for 36 h. Cells were then treated with 50 µg/mL cycloheximide (CHX) for an indicated time interval. Cell lysates were subjected to Western blot analyses. The EGFR protein levels were quantified and a plot representing protein half-life was presented. i H292 or H1975 cells stably expressing HA-FBXL2 or HA-FBXL2C420S were subjected to Western blot analyses. j H1299 or H1975 cells stably expressing HA-FBXL2, HA-FBXL2C420S, or a vector control were treated with MG132 for 4 h before harvesting for IP-Western analyses. k H1299 or H1975 cells stably expressing HA-FBXL2 or HA-FBXL2C420S were subjected to fractionation of cytoplasmic (CYTO) and plasma membrane (PM), followed by Western blot analyses. l H1299 cells stably expressing Flag-FBXL2 or Flag-FBXL2C420S were treated with MG132 for 6 h prior to immunostaining for Flag (Red) and endogenous EGFR (Green) and counterstained with DAPI. Scale bar = 25 µm. The experiment was repeated three times independently with similar results (b, d, f, g, and i−l). Source data are provided as a Source data file.