anti pegfr tyr1038  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: B7-H3-mediated cis-inhibition of EGFR by a tumor-selective bispecific antibody enhances anti-tumor efficacy and minimizes toxicities

doi: 10.1038/s41467-026-69703-7

Figure Lengend Snippet: a Screening workflow schematic. *7B6 binding affinity was below the detection limit. b In vitro efficacy and safety validation. Efficacy: IC 50 ratio of monovalent EGFR mAb over B7-H3/EGFR bsAb in PC9-B7-H3 cells. Safety: IC 50 ratio of B7-H3/EGFR bsAb in A431 skin cells vs PC9-B7-H3. The IC 50 values for B7-H3/EGFR bsAb are the means from three biological replicates. c Growth inhibition assays of NCI-H358 and A431 cell lines ( n = 3 biological replicates). d Competitive EGFR binding assay. NCI-H358 cells pre-incubated with fluorescently labeled Zalutumumab (Zalu) were treated with 20G5/Zalu bsAb or Zalu ( n = 3 biological replicates). e EGF receptor occupancy assay. Cells treated with 20G5/Zalu or Zalu were stained with AF647-Zalu to quantify unoccupied EGFR ( n = 3 biological replicates). f EGFR internalization. Cells were treated with 5 nM IBI334, Zalu, or Null/Zalu and surface EGFR levels were detected using a non-competing anti-EGFR antibody ( n = 3 biological replicates). g Western blot of EGFR pathway-related proteins in NCI-H358 cells treated with 20G5/Zalu, Zalu, Null/Zalu or combo (20G5/null + Null/Zalu) ( n = 2 biological replicates). h Time-course analysis of EGFR signaling inhibition by Western blot (left, n = 2 biological replicates), and quantitated band intensities (left). i , j Ligand blockade assays. Flow cytometry (left) measuring EGF ( i ) or TGF-α ( j ) binding after treatment ( n = 3 biological replicates). EGFR signaling pathway was analyzed by Western blot (right, n = 2 biological replicates). k – m Cis-inhibition experiment. Cells were seeded at low-density (3 × 10⁵ cells/10 cm²) to prevent cell-cell contact. l Western blots of WT SK-MES-1 vs B7-H3-overexpressing (B7-H3 OE ) SK-MES1 treated with indicated antibodies for 4 h ( n = 2 biological replicates). (m) Western blot of SK-MES-1 B7-H3 OE cells ( n = 2 biological replicates). n Cis- vs trans-inhibition. Fluorescence microscopy of pEGFR (red) in SK-MES-1 (white arrows) and GFP + SK-MES-1-B7-H3 OE (green) co-cultures with/without 1 nM 20G5/Zalu ( n = 3 biological replicates). Schematic (left) and representative images (right) are shown. For EGFR internalization ( f ), statistical significance was assessed by two-way ANOVA (Tukey’s post hoc test). Data bars are mean ± SD. Source data are provided as a Source Data file.

Article Snippet: For IHC, multiplexed immunofluorescence (mIF), and western blotting (WB), the following antibodies were utilized: EGFR (CST, 4267), pEGFR (CST, 3777), B7-H3 (CST, 14508), AKT (CST, 4691), pAKT (CST, 4060), ERK (CST, 4695), pERK (CST, 4370), S6 (CST, 2217S), pS6 (CST, 2211S), GAPDH (CST, 2218), Pan CK (Gene Tech, GM351514 ), and Ki67 (CST, 9027S).

Techniques: Binding Assay, In Vitro, Biomarker Discovery, Inhibition, Incubation, Labeling, Staining, Western Blot, Flow Cytometry, Fluorescence, Microscopy

Journal: mBio

Article Title: Candida albicans -induced ubiquitination of EGFR reveals novel host–fungal interaction pathways

doi: 10.1128/mbio.03448-25

Figure Lengend Snippet: C. albicans induces EGFR phosphorylation at Y1045 and EGFR ubiquitination in TR146 oral epithelial cells in an ECE1 -driven manner. ( A ) Schematic of EGFR phosphorylation and ubiquitination. Created with BioRender.com. ( B ) TR146 cells were infected with wild-type (WT) C. albicans (BWP17), an ECE1 null strain ( ece1 Δ/Δ), an ECE1 re-integrant strain ( ece1 Δ/Δ + ECE1 ), or a candidalysin null strain ( ece1 Δ/Δ + ECE1 Δ184–279 ) for 4 h at an MOI of 10 or stimulated with PBS as a vehicle control. Lysates (10 µg) were electrophoresed on gradient gels to detect pEGFR Y1045 and α-actin via Western blot. Data are representative of three independent experiments. ( C ) TR146 cells were infected with WT C. albicans (SC5314), ece1 Δ/Δ, ece1 Δ/Δ + ECE1 , ece1 Δ/Δ+ ECE1 Δ184–279 , and als3 Δ/Δ for 6 h at an MOI of 5 or stimulated with PBS as a vehicle control. EGF 100 ng/mL was used as a positive control. Lysates were collected and EGFR was immunoprecipitated. Immunoprecipitation (IP) samples were electrophoresed on gradient gels to detect total EGFR and ubiquitin via Western blot. Data are representative of three independent experiments. Dashed vertical lines indicate omitted, extraneous portions of blot images. ( D ) Relative protein levels in panel C were quantified using densitometry. Ubiquitin was normalized to total EGFR and expressed relative to the control (PBS), which was set to 1. Data represent the average of three to seven independent experiments and are presented as mean values with standard error of the mean (SEM) error bars. Statistical significance was determined using the Kruskal–Wallis test with Dunn’s multiple comparison test. P < 0.05, *** P < 0.001. ( E ) TR146 cells were infected with WT C. albicans (SC5314), ece1 Δ/Δ, and als3 Δ/Δ for 6 h at an MOI of 5 or stimulated with PBS as a vehicle control. Lysates were collected and EGFR was immunoprecipitated. IP samples were electrophoresed on gradient gels to detect total EGFR, Grb2, AP2M1, and HRS via Western blot. Dashed vertical lines indicate omitted, extraneous portions of blot images. Data are representative of three independent experiments. ( F–H ) Relative protein levels in panel E were quantified using densitometry. Grb2, AP2M1, and HRS were normalized to total EGFR and expressed relative to the control (PBS), which was set to 1. Data represent the average of three to four independent experiments and are presented as mean values with SEM error bars. Statistical significance was determined using a parametric test (ordinary one-way ANOVA with Dunnett’s multiple comparison test). * P < 0.05. ns, not significant.

Article Snippet: pEGFR Y1045 , Rabbit , 1:1,000 , Cell Signaling Technology , 2237.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Infection, Control, Western Blot, Positive Control, Immunoprecipitation, Comparison