Journal: Cell reports
Article Title: Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR
doi: 10.1016/j.celrep.2022.110950
Figure Lengend Snippet: (A) Schematics of the proximity labeling TMTplex11 experiment in HCT116 and HEK293T cells. Serum-starved cells were either untreated (control) or treated with 100 ng/mL EGF for 2–10 min to analyze the proximal proteins during receptor activation and internalization. Biological replicates (individually grown cell cultures) were included except for 10 min treatment. (B and C) Time course of the relative intensity of TGF compared with LSR (plasma membrane) and Sec13 (ERES) proximal to EGFR-APEX2 in HCT116 (B) and HEK293T (C) cells stimulated with EGF in the experiment described in (A). (D–F) HCT116/EGFR-APEX2 (D), HSC3 (E), and HeLa/FAP-EGFR (F) cells were stimulated with 10 ng/mL EGF-Rh for 15 min and fixed and labeled with TFG antibodies. Three-dimensional (3D) confocal images were acquired through 561- (magenta, EGF-Rh) and 488-nm (green, TFG) channels. (G) Quantification of the fraction of TFG co-localized with EGF-Rh from images exemplified in (E) and (F). Bar graph represents mean values with SDs (n of 13–32 fields of view [FOVs] from 2 to 5 independent experiments). (H) HSC3 cells transiently expressing GFP-TFG were stimulated with 10 ng/mL EGF-Rh for 15 min. 3D confocal images were acquired through 561- (magenta, EGF-Rh) and 488-nm (Green, GFP-TFG) channels. (I) Quantification of the fraction of EGF-Rh co-localized with GFP-TFG from images exemplified in (H). Bar graph represents mean values with SDs (n = 11 individual cells) from 2 independent experiments. All images are single sections through the middle of the cell of representative 3D images. Insets in (D)–(F) represent enlargements of the regions indicated by rectangles. Arrows indicate examples of co-localization. Asterisks show positions of nuclei. Scale bars, 10 μm in full images and 5 μm in insets.
Article Snippet: Mouse receptor grade EGF (Corning) was 125 I-conjugated, and 125 I-EGF internalization, recycling and degradation assays were performed as described ( ).
Techniques: Labeling, Control, Activation Assay, Clinical Proteomics, Membrane, Expressing
![(A) Schematics of the proximity labeling TMTplex11 experiment in HCT116 and HEK293T cells. Serum-starved cells were either untreated (control) or treated with 100 ng/mL <t>EGF</t> for 2–10 min to analyze the proximal proteins <t>during</t> <t>receptor</t> activation and internalization. Biological replicates (individually grown cell cultures) were included except for 10 min treatment. (B and C) Time course of the relative intensity of TGF compared with LSR (plasma membrane) and Sec13 (ERES) proximal to EGFR-APEX2 in HCT116 (B) and HEK293T (C) cells stimulated with EGF in the experiment described in (A). (D–F) HCT116/EGFR-APEX2 (D), HSC3 (E), and HeLa/FAP-EGFR (F) cells were stimulated with 10 ng/mL EGF-Rh for 15 min and fixed and labeled with TFG antibodies. Three-dimensional (3D) confocal images were acquired through 561- (magenta, EGF-Rh) and 488-nm (green, TFG) channels. (G) Quantification of the fraction of TFG co-localized with EGF-Rh from images exemplified in (E) and (F). Bar graph represents mean values with SDs (n of 13–32 fields of view [FOVs] from 2 to 5 independent experiments). (H) HSC3 cells transiently expressing GFP-TFG were stimulated with 10 ng/mL EGF-Rh for 15 min. 3D confocal images were acquired through 561- (magenta, EGF-Rh) and 488-nm (Green, GFP-TFG) channels. (I) Quantification of the fraction of EGF-Rh co-localized with GFP-TFG from images exemplified in (H). Bar graph represents mean values with SDs (n = 11 individual cells) from 2 independent experiments. All images are single sections through the middle of the cell of representative 3D images. Insets in (D)–(F) represent enlargements of the regions indicated by rectangles. Arrows indicate examples of co-localization. Asterisks show positions of nuclei. Scale bars, 10 μm in full images and 5 μm in insets.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8364/pmc09248364/pmc09248364__nihms-1816591-f0005.jpg)
