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

Journal:

Article Title: Grb2 Regulates Internalization of EGF Receptors through Clathrin-coated Pits

doi: 10.1091/mbc.E02-08-0532

Figure Lengend Snippet: Schematic representation of EGF receptor mutants. (A) Wild-type EGFR (WT) is drawn with the extracellular, transmembrane (TM) and intracellular domain (kinase and C terminus). Main tyrosine phosphorylation sites (residues 992, 1068, 1086, 1148, and 1173) and a Cbl binding site (Tyr1045) are indicated in WT receptor. Mutants have C-terminal truncation of 79 (C′1107), 96 (C′1090), 114 (C′1072), 123 (C′1063), and 164 amino acid residues (C′1022). Tyrosine substitutions by phenylalanines (F) are indicated. (B) To confirm the correct size of truncated EGFR mutants, PAE cells expressing WT, C′1107, C′1090, C′1072, C′1063, or C′1022 were lysed, and the EGFR was detected in lysates by immunoblotting with antibody 2913.

Article Snippet: Internalization of 125 I-EGF and 125 I-transferrin Mouse receptor-grade EGF was obtained from Collaborative Research Inc. (Bedford, MA) and iodinated using a modified chloramine T method as described previously ( Sorkin et al. , 1991 ).

Techniques: Binding Assay, Expressing, Western Blot

Journal:

Article Title: Human Herpesvirus 8 Envelope Glycoprotein B Mediates Cell Adhesion via Its RGD Sequence

doi: 10.1128/JVI.77.5.3131-3147.2003

Figure Lengend Snippet: HHV-8-gBΔTM activates the integrin-dependent FAK components. Serum-starved HFFs in chamber slides were incubated with 200 μl of serum-free DMEM (mock) or LPA (20 ng/ml) or gBΔTM protein (50 ng/ml) in serum-free DMEM or gBΔTM-RGA (50 ng/ml) in serum-free DMEM. After incubation for 5 min at 37°C, cells were washed, fixed, permeabilized, and reacted with rabbit anti-p125FAK and mouse MAb to paxillin, followed by anti-rabbit-tetramethyl rhodamine isothiocyanate and anti-mouse-FITC antibodies. Stained cells were examined under a fluorescence microscope with appropriate filters. The arrowheads indicate representative areas of FAK-paxillin colocalization. Magnification, ×1,000.

Article Snippet: Mouse anti-phospho-FAK antibody (Y397, BD) and mouse MAb to paxillin (clone 177) were from Transduction Laboratories, Los Angeles, Calif. 125 I-labeled epidermal growth factor (EGF) was from ICN, Irvine, Calif.

Techniques: Incubation, Staining, Fluorescence, Microscopy