grb2 3972s  (Cell Signaling Technology Inc)

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: GRB2 , Rabbit , 1:1,000 , Cell Signaling Technology , 3972.

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

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 promotes EGFR degradation through Ece1p and Als3p, predominantly mediated by the lysosomal pathway, and induces the recruitment of adaptor proteins Grb2, AP2M1, and HRS. ( A–C ) TR146 cells were infected with different C. albicans strains (wild-type [WT] SC5314, ece1 Δ/Δ and als3 Δ/Δ) or stimulated with PBS as a vehicle control for 2 h (MOI 10), 8 h (MOI 1), and 12 h (MOI 1). Cell lysates were electrophoresed on gradient gels to detect total EGFR and α-actin via Western blot. Data are representative of three independent experiments. ( D and E ) TR146 cells were pre-treated for 1 h with 0.25 µM of lysosomal inhibitor Bafilomycin A1 (BafA1) and 10 µM of proteasomal inhibitor MG-132 or DMSO as a vehicle control, and then treated with 100 ng/mL EGF as a positive control for 1 h or infected with SC5314 (MOI 5) for 10 h. Cell lysates were electrophoresed on gradient gels to detect total EGFR and α-actin via Western blot. Data are representative of three independent experiments. Dashed vertical lines indicate omitted, extraneous portions of blot images. ( F and G ) Relative protein levels in panels D and E were quantified using densitometry. Total EGFR was normalized to α-actin and expressed relative to the control (DMSO + water or DMSO + PBS), which was set to 100. Data are presented as mean values with standard error of the mean 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: GRB2 , Rabbit , 1:1,000 , Cell Signaling Technology , 3972.

Techniques: Infection, Control, Western Blot, Positive Control, Comparison

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 dynamically regulates Egfr , ubiquitin pathway-associated genes, and host protein ubiquitination in an oropharyngeal candidiasis mouse model. C57BL/6J mice were sublingually infected with wild-type (WT) C. albicans AHY940 and mutant strains ece1 Δ/Δ and als3 Δ/Δ, and tongues were harvested at 24 and 48 h post-infection. Gene expression of ( A ) Egfr , ( F ) Tnfaip3 , ( G ) Usp36 , and ( H ) Usp13 was quantified using ΔΔCt relative to the naïve control. Data were collected from one to three independent experiments with three to four mice/group. Data are plotted as box plots, where the central line represents the median; the box indicates the interquartile range; and the whiskers extend to the minimum and maximum values. Individual data points represent biological replicates. A Shapiro–Wilk test was first used to determine data normality. If data were normal, a parametric test (ordinary one-way ANOVA with Dunnett’s multiple comparison test) was performed. If data were not normal, a non-parametric test (Kruskal–Wallis test with Dunn’s multiple comparison test) was used to determine statistical significance. An unpaired t -test was used for pairwise comparisons between specific strains. ( B ) Tongue lysates were electrophoresed on gradient gels and analyzed by Western blot to detect the presence of ubiquitin and Grb2. α-Actin was used as a loading control. Data are representative of three independent experiments. ( C–E ) Relative protein levels in panel B were quantified using densitometry. Target protein levels were normalized to actin and expressed relative to the naïve control, which was set to 1. Data are presented as median with interquartile range. Individual data points represent biological replicates. Statistical significance was determined using an unpaired non-parametric t -test (Kolmogorov–Smirnov test). * P < 0.05, ** P < 0.01, **** P < 0.0001. ns, not significant.

Article Snippet: GRB2 , Rabbit , 1:1,000 , Cell Signaling Technology , 3972.

Techniques: Ubiquitin Proteomics, Infection, Mutagenesis, Gene Expression, Control, Comparison, Western Blot

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: Model for epithelial cell activation and EGFR trafficking induced by C. albicans . (A) During epithelial infection, C. albicans hyphae form an invasion pocket, predominantly through the binding of the fungal adhesin/invasin Als3p to the EGFR/HER2 complex on epithelial cells. Hyphae secrete the cytolytic peptide toxin candidalysin, which accumulates in the invasion pocket prior to forming pores in the plasma membrane, triggering calcium influx and cell damage (LDH release). Calcium influx stimulates the activation of matrix metalloproteinases, which release surface-tethered epidermal growth factor receptor (EGFR) ligands, epigen (EPGN), epiregulin (EREG), and amphiregulin (AREG). (B) These ligands bind to EGFR, triggering receptor dimerization and activation. (C) EGFR activation induces ERK1/2 phosphorylation and the subsequent activation of the transcription factor c-Fos. This leads to the expression of cytokines (G-CSF, GM-CSF, IL-1α, and IL-1β), which are released from the cell, inducing neutrophil recruitment and type 17 immunity. (D) In parallel, candidalysin activates the p38-MAPK pathway, which can also activate EGFR and Hsp27 and controls IL-6 secretion. (E) Candidalysin also drives the upregulation of ubiquitin C ( UBC ) and ubiquitin pathway-associated genes such as USPs and TNFAIP3 , as well as an increase in total protein ubiquitination. (F) Once EGFR is activated, adaptor protein Grb2 is recruited, and EGFR is ubiquitinated. (G) EGFR is then internalized into early endosomes, trafficked into multivesicular bodies (MVBs), and finally degraded in lysosomes. The degradation of EGFR negatively regulates EGFR signaling. This model is based on in vitro data. Figure created with BioRender.com .

Article Snippet: GRB2 , Rabbit , 1:1,000 , Cell Signaling Technology , 3972.

Techniques: Activation Assay, Infection, Binding Assay, Clinical Proteomics, Membrane, Phospho-proteomics, Expressing, Ubiquitin Proteomics, In Vitro