Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 negatively regulated IFN-β induced by H13N2 infection. (a, B) luciferase reporter assays were used to assess the impact of FGF8 overexpression on IFN-β and ISRE promoter activity in A549 cells infected with H13N2 at an MOI of 1. (C-F) FGF8-overexpressing A549 cells were infected with H13N2 at an MOI of 1. At 12 hours post-infection (hpi), IFN-β levels in the cell supernatant were measured using ELISA (C), and IFN-β mRNA levels were evaluated by RT-qPCR (d). At 24 hpi, the mRNA levels of interferon-stimulated genes MX1 (e) and IFIT1 (f) were assessed by RT-qPCR. (G-J) stable FGF8-knockdown A549 cells were infected with H13N2 at an MOI of 1. At 12 hpi, IFN-β levels in the cell supernatant were quantified by ELISA (G), and IFN-β mRNA levels were evaluated using RT-qPCR (H). At 24 hpi, the mRNA levels of MX1 (i) and IFIT1 (J) were assessed by RT-qPCR. (K and L) Western blot analysis evaluated RIG-I, p-TBK1, and p-IRF3 expression in A549 cells with FGF8 overexpression (L) or knockdown (K) at 12 hours after H13N2 infection (MOI = 1). Band intensities were quantified by densitometric analysis. Statistical analysis was performed using two-tailed unpaired Student’s t-tests, with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Infection, Luciferase, Over Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 drives ubiquitin – proteasomal degradation of RIG-I. (a) FGF8 inhibits RIG-I-mediated signaling. A luciferase reporter assay was performed to evaluate the effect of FGF8 overexpression on IFN-β promoter activation induced by RIG-I. (B and C) FGF8 does not affect RIG-I transcription. RIG-I mRNA levels were quantified by RT-qPCR in FGF8-overexpressing A549 cells at 0, 6, and 12 hours post-infection with H13N2 (b) or H1N1 (C) at an MOI of 1. (d) dose-dependent reduction of RIG-I protein. A549 cells were transfected with increasing amounts of Flag-FGF8 plasmid for 24 hours, followed by infection with H13N2 (MOI = 1) for 12 hours. RIG-I protein levels were analyzed by Western blot, and band intensities were quantified by densitometry. (e) FGF8 reduces RIG-I stability. FGF8-overexpressing A549 cells were infected with H13N2 (MOI = 1) and treated with cycloheximide (CHX, 50 µg/mL) for the indicated time periods. Protein levels were analyzed by Western blot, and the relative abundance of HA-RIG-I was quantified to assess protein degradation rates. (F and G) proteasome inhibition restores RIG-I levels. A549 cells infected with H13N2 (f) or H1N1 (G) at an MOI of 1 were treated with DMSO, chloroquine (CQ, 50 µM), or MG132 (10 µM) for 6 hours. RIG-I expression was analyzed by Western blot, with relative protein levels quantified by densitometry. (H and I) FGF8 promotes K48-linked ubiquitination of RIG-I. HEK-293T cells were co-transfected with the indicated plasmids and treated with MG132 for 6 hours. (H) Total ubiquitination of RIG-I was assessed by immunoprecipitation with anti-HA antibody followed by immunoblotting (ib) with anti-Myc. (i) K48- or K63-linked ubiquitination was analyzed using specific ubiquitin mutants. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Luciferase, Reporter Assay, Over Expression, Activation Assay, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Western Blot, Inhibition, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: TRIM16 mediated RIG-I degradation and promoted influenza virus replication. (a) Co-immunoprecipitation analysis was performed in cells transfected with Flag-TRIM16 and HA-RIG-I, with or without H13N2 infection (MOI = 1), to verify the interaction. (b) immunofluorescence microscopy showing the localization of TRIM16 (green) and RIG-I (red) in cells infected with H13N2 or mock-infected (NC). Nuclei were stained with DAPI (blue). Note that TRIM16 and RIG-I show diffuse distribution in the NC group but form co-localized puncta (yellow) upon H13N2 infection. Scale bar: 5 μm. (C) in vitro ubiquitination assay to verify the direct E3 ligase activity of TRIM16 using wt and ΔB-Box mutant proteins. (d) in vitro ubiquitination assay to determine the linkage specificity of TRIM16-mediated RIG-I ubiquitination using K48-only and K63-only ubiquitin mutants. (e) bioinformatic analysis using PONDR revealed the presence of intrinsically disordered regions (IDRs) in the FGF8 protein sequence. (f) fluorescence microscopy of A549 cells transfected with EGFP-FGF8 (green). Nuclei were stained with DAPI. Scale bar represents 10 μm. (G) TurboID-based proximity labeling assay was performed in cells expressing FGF8-TurboID. Biotinylated proteins were captured using streptavidin beads, and the pulled-down proteins were analyzed by Western blot to detect the presence of RIG-I and TRIM16. (H and I) validation of TRIM16 knockdown. RT-qPCR (H) and Western blot (i) confirmed the silencing efficiency in A549 cells. (J) control and TRIM16-silenced A549 cells were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein levels (NP, PB1, PB2) were analyzed by Western blot, and band intensities were quantified by densitometry. (K) RT-qPCR analysis of IFN-β mRNA levels in TRIM16-silenced A549 cells 12 hours post-infection with H13N2 (MOI = 1). (L) Western blot confirmation of TRIM16 overexpression (OE-TRIM16). (M) A549 cells overexpressing TRIM16 were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein expression was analyzed by Western blot and quantified by densitometry. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Virus, Immunoprecipitation, Transfection, Infection, Immunofluorescence, Microscopy, Staining, In Vitro, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Sequencing, Fluorescence, Labeling, Expressing, Western Blot, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Control, Over Expression, Two Tailed Test

Journal: iScience

Article Title: UBE2M-mediated EGFR neddylation drives keratinocyte proliferation in psoriasis

doi: 10.1016/j.isci.2026.115784

Figure Lengend Snippet: UBE2M is upregulated in human psoriatic skin and IMQ-induced psoriasis-like dermatitis in mice (A) Western blot analysis of Nedd8 protein expression in healthy skin samples and psoriatic lesional skin samples ( n = 5 human per each group). High-molecular-weight NEDD8-protein conjugates were used as a readout of global neddylation. The band at ∼10 kDa corresponds to free NEDD8. GAPDH served as the loading control. (B) Western blot analysis and quantification of UBE2M protein expression in healthy skin samples (HD) and psoriatic lesional skin samples (PL, n = 5 human per each group). GAPDH was used as the loading control. (C) The expression of UBE2M in psoriatic lesions compared with healthy skin from GEO: GSE13355 . UBE2M is upregulated in psoriatic lesions. (D) mRNA expression level of UBE2M was quantified in HD and PL ( n = 5 human per each group) via quantitative PCR. β-actin served as the internal control. (E) Immunohistochemical detection of UBE2M expression in healthy and psoriatic lesional skin ( n = 5 human per each group). Enlarged images in the box were present on the side below. Scale bars, 100 μm. (F) A diagram of a psoriasis model in C57BL/6, prepared by IMQ. (G) mRNA expression level of UBE2M was quantified in VAS and IMQ group ( n = 4 mice per each group) via quantitative PCR. β-actin served as the internal control. (H) Immunohistochemical detection of UBE2M expression in the VAS and IMQ group ( n = 4 mice per each group). Enlarged images in the box were present on the side below. Scale bars, 100 μm. (I) Western blot analysis and quantification of UBE2M protein expression in the VAS group and IMQ group ( n = 4 mice per each group). GAPDH was employed as the loading control. (J) Western blot analysis of Nedd8 protein expression in the VAS and IMQ groups ( n = 4 mice per each group). High-molecular-weight NEDD8-protein conjugates were used as a readout of global neddylation. The band at ∼10 kDa corresponds to free NEDD8. GAPDH served as the loading control. (K) Western blot analysis and quantification of UBE2M protein expression in HaCaT cells treated with the indicated concentration. ( n = 3 biologically independent experiments). (L) Western blot analysis was performed to evaluate UBE2M protein levels in HaCaT cells exposed to IL-17A (25 ng/mL) for the specified durations, with GAPDH serving as the loading control. ( n = 3 biologically independent experiments). All data are expressed as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (B, D, E, G, H, and I) or by one-way ANOVA with post hoc Tukey (K and L).

Article Snippet: For co-immunoprecipitation of UBE2M, NEDD8, or EGFR, protein A/G magnetic beads (MedChemExpress, Cat# HY-K0202) were pre-incubated with the indicated antibodies (5 μL per reaction) at 4°C for 6 h, followed by overnight incubation with lysates derived from one 10-cm culture dish per sample.

Techniques: Western Blot, Expressing, High Molecular Weight, Control, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Concentration Assay

Journal: iScience

Article Title: UBE2M-mediated EGFR neddylation drives keratinocyte proliferation in psoriasis

doi: 10.1016/j.isci.2026.115784

Figure Lengend Snippet: UBE2M regulates KC proliferation and migration (A) Western blot analysis was conducted to assess UBE2M expression in HaCaT cells treated with OE-Control or OE-UBE2M. GAPDH served as the loading control. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (B) Western blot analysis was performed to evaluate UBE2M expression in HaCaT cells transfected with sh-NC or sh-UBE2M. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (C–E) Cell proliferation in HaCaT, ker-CT, and NHEK cells with OE-UBE2M and sh-UBE2M expression was assessed using the CCK-8 assay. ( n = 3 biologically independent experiments). (F) Ki67 staining was performed to assess cell proliferation in NHEK cells with UBE2M overexpression and knockdown. Nuclei were visualized using DAPI staining (blue). The percentage of Ki67-positive cells was quantified. Scale bars, 50 μm. ( n = 3 biologically independent experiments). (G) EdU staining was used to evaluate cell proliferation in NHEK cells with UBE2M overexpression and knockdown. Nuclei were stained with DAPI (blue) for visualization. The proportion of EdU-positive cells was calculated. Scale bars, 50 μm ( n = 3 biologically independent experiments). (H) Representative image of a colony formation assay showing HaCaT cell growth after treatment with OE-UBE2M or sh-UBE2M. The number of colonies of each group was quantified. ( n = 3 biologically independent experiments). (I) Wound-healing assay to analyze the migration capability of HaCaT cells with OE-UBE2M and sh-UBE2M mutation expression. Cells were photographed every 24 h. The migration rates of each group were quantified. Scale bars, 500 μm ( n = 3 biologically independent experiments). (J) CCK-8 assay analysis of cell proliferation of HaCaT cells with IL-17 upon UBE2M knockdown. ( n = 3 biologically independent experiments). (K) CCK-8 assay analysis of cell proliferation of HaCaT cells with mln4924 upon UBE2M overexpression. ( n = 3 biologically independent experiments). All data are expressed as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (C, D, E, J, and K) or by one-way ANOVA with post hoc Tukey (F, G, H, and I).

Article Snippet: For co-immunoprecipitation of UBE2M, NEDD8, or EGFR, protein A/G magnetic beads (MedChemExpress, Cat# HY-K0202) were pre-incubated with the indicated antibodies (5 μL per reaction) at 4°C for 6 h, followed by overnight incubation with lysates derived from one 10-cm culture dish per sample.

Techniques: Migration, Western Blot, Expressing, Control, Transfection, CCK-8 Assay, Staining, Over Expression, Knockdown, Colony Assay, Wound Healing Assay, Mutagenesis

Journal: iScience

Article Title: UBE2M-mediated EGFR neddylation drives keratinocyte proliferation in psoriasis

doi: 10.1016/j.isci.2026.115784

Figure Lengend Snippet: UBE2M knockdown inhibits the immunopathological changes and blocks psoriasis development in the IMQ-induced mouse model (A) mRNA expression level of UBE2M was quantified in WT and Ube2m +/− mice via quantitative PCR. ( n = 4 mice per each group). β-actin served as the internal control. (B) UBE2M expression in the skin of WT nd Ube2m +/− mice was detected by Western blot and quantification for the indicated bands. ( n = 4 mice per each group). GAPDH served as the loading control. (C) Representative photos of dorsal skin in WT and Ube2m +/− mice with IMQ and VAS treatments. Images from all biological replicates ( n = 4 mice per group) are provided in . (D) Representative photos of spleen in WT and Ube2m +/− mice with IMQ treatments ( n = 4 mice per each group). Mouse spleen index in WT and Ube2m +/− mice after IMQ treatment and quantification for the spleen index. Images from all biological replicates ( n = 4 mice per group) are provided in . (E) Scores of erythema, scaling, thickness, and cumulative scores of WT mice and Ube2m +/− mice after IMQ treatment. ( n = 4 mice per each group). (F) Histological staining of skin tissue with H&E from the back of WT and Ube2m +/− mice. Enlarged images in the box were present on the lower side. Scale bars, 100 μm ( n = 4 mice per each group). (G) Immunohistochemistry staining of IMQ or VAS-treated skin of WT and Ube2m +/− mice by Ki-67. The quantitative analysis of Ki-67 positive cell rates was detected by ImageJ. Scale bars, 100 μm ( n = 4 mice per each group). (H–K) Quantitative PCR analysis of mRNA level of the Il17a , Il23 , Tnfα , and MCP-1 genes in WT mice and Ube2m +/− mice after IMQ or VAS treatment. ( n = 4 mice per each group). β-actin served as the internal control. All data are expressed as mean ± SD. ns., not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (A, B, D, and E) or by one-way ANOVA with post hoc Tukey (F, G, H, I, J, and K).

Article Snippet: For co-immunoprecipitation of UBE2M, NEDD8, or EGFR, protein A/G magnetic beads (MedChemExpress, Cat# HY-K0202) were pre-incubated with the indicated antibodies (5 μL per reaction) at 4°C for 6 h, followed by overnight incubation with lysates derived from one 10-cm culture dish per sample.

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining, Immunohistochemistry

Journal: iScience

Article Title: UBE2M-mediated EGFR neddylation drives keratinocyte proliferation in psoriasis

doi: 10.1016/j.isci.2026.115784

Figure Lengend Snippet: UBE2M modulates the expression and activity of EGFR through direct interaction with EGFR (A) EGFR and p-EGFR were modified upon the overexpression of UBE2M in HaCaT cell lines detected by western blot, alongside statistical analysis of the indicated proteins. ( n = 3 biologically independent experiments). GAPDH served as the loading control. (B) Densitometric gray value analysis of the p-EGFR/EGFR ratio was performed using ImageJ software, with GAPDH serving as the loading control. ( n = 3 biologically independent experiments). (C) EGFR was modified upon the knockdown of UBE2M in HaCaT cell lines detected by western blot. ( n = 3 biologically independent experiments). GAPDH served as the loading control. (D) HaCaT cells were transfected with UBE2M or UBE2M siRNA and then treated in the presence of cycloheximide (2 μg/ml) for the indicated times at 37°C. Then, it was detected by western blot. ( n = 3 biologically independent experiments). (E) HA-tagged ubiquitin and Flag-tagged EGFR were co-expressed in cells with or without UBE2M. EGFR was immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-ubiquitin antibody. UBE2M overexpression decreased EGFR ubiquitination. Input shows the expression of HA-Ub, Flag-EGFR, and UBE2M. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (F) EGFR expression in the skin of WT and Ube2m +/− mice was evaluated by western blot, with GAPDH serving as the loading control and quantification for the indicated bands. ( n = 4 mice per each group). (G) Immunohistochemical staining of EGFR in dorsal skin from wild-type and Ube2m heterozygous mice treated with VAS or IMQ. Scale bars, 100 μm ( n = 4 mice per each group). (H) Ligand-induced dimerization assay of EGFR by western blot. GAPDH serves as the loading control. ( n = 3 biologically independent experiments). (I) Western blot analysis of the activation of EGFR and AKT in HaCaT cells in the presence or absence of sh-UBE2M with or without EGF (20 ng/ml) stimulation. Densitometric gray value quantification of p-EGFR/EGFR ratios was performed using ImageJ software, with GAPDH serving as the loading control. ( n = 3 biologically independent experiments). (J) Protein expression of EGFR was examined by immunofluorescence staining in HaCaT cells treated with UBE2M. Scale bars, 10 μm. Representative images from one of three independent experiments are shown. ( n = 3 independent repeat experiments). (K–L) The interaction of UBE2M and EGFR was detected by CoIP. HaCaT cells were transfected with Flag-EGFR and HA-UBE2M for 48 h, followed by anti-HA (K) or anti-Flag (L) immunoprecipitation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (M) Endogenous UBE2M in HaCaT cells was shown to interact with EGFR through co-immunoprecipitation using UBE2M antibody. Mouse IgG served as a control. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (N) Co-immunoprecipitation with EGFR antibody demonstrated the interaction between endogenous EGFR and UBE2M in HaCaT cells. Mouse IgG was used as a control. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (O) In mouse skin, endogenous UBE2M interacted with EGFR through co-immunoprecipitation using UBE2M antibody. Mouse IgG acted as a control. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (P) Co-immunoprecipitation with EGFR antibody revealed the interaction between endogenous EGFR and UBE2M in mouse skin. Mouse IgG was utilized as a control. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (Q) The proliferation of HaCaT cells after overexpression of UBE2M and stimulation or non-stimulation of AG1478 was analyzed by the CCK-8 method. ( n = 3 biologically independent experiments). All data are expressed as mean ± SD. ns., not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 by unpaired Student’s t test (A, B, D, F, and Q) or by one-way ANOVA with post hoc Tukey (C, G, H, and I).

Article Snippet: For co-immunoprecipitation of UBE2M, NEDD8, or EGFR, protein A/G magnetic beads (MedChemExpress, Cat# HY-K0202) were pre-incubated with the indicated antibodies (5 μL per reaction) at 4°C for 6 h, followed by overnight incubation with lysates derived from one 10-cm culture dish per sample.

Techniques: Expressing, Activity Assay, Modification, Over Expression, Western Blot, Control, Software, Knockdown, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Immunohistochemical staining, Staining, Activation Assay, Immunofluorescence, CCK-8 Assay

Journal: iScience

Article Title: UBE2M-mediated EGFR neddylation drives keratinocyte proliferation in psoriasis

doi: 10.1016/j.isci.2026.115784

Figure Lengend Snippet: UBE2M Enhances EGFR Neddylation (A) The NAE inhibitor MLN4924 inhibited the increase in EGFR expression mediated by UBE2M in the HaCaT cell line. The cells were administered MLN4924 (0.3 μM, 12 h) before harvesting. GAPDH served as the loading control and quantification for the indicated bands. ( n = 3 biologically independent experiments). (B) Western blot analysis of EGFR expression in HaCaT cells treated with UBE2M or UBE2M-C111S. GAPDH served as the loading control and quantification for the indicated bands. ( n = 3 biologically independent experiments). (C) EGFR associates with NEDD8. HA-NEDD8 and Flag-EGFR were transfected into HaCaT cells and treated with 0.3 μM MLN4924 for 12 h. Cell lysis was followed by immunoprecipitation with a Flag antibody, followed by immunoblotting with the indicated antibodies. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (D) Endogenous EGFR is associated with NEDD8 via co-immunoprecipitation with the EGFR antibody. Mouse IgG was utilized as a control. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (E) The neddylation of EGFR is increased upon UBE2M overexpression in HaCaT cells, detected by immunoprecipitation with a Flag antibody. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (F) The neddylation of EGFR is reduced upon UBE2M knockdown in HaCaT cells, detected by immunoprecipitation with a Flag antibody. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (G) In HaCaT cells, endogenous EGFR was shown to interact with NEDD8 through co-immunoprecipitation using EGFR antibody following IL-17 stimulation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (H) In HaCaT cells, endogenous NEDD8 was found to associate with EGFR through co-immunoprecipitation with NEDD8 antibody after IL-17 stimulation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (I) In Ker-CT cells, endogenous EGFR interacted with NEDD8 via co-immunoprecipitation using EGFR antibody following IL-17 stimulation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (J) Likewise, endogenous NEDD8 in Ker-CT cells was associated with EGFR through co-immunoprecipitation with NEDD8 antibody after IL-17 stimulation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (K) Endogenous EGFR in NHEK cells was found to associate with NEDD8 via co-immunoprecipitation using EGFR antibody after IL-17 treatment. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (L) In NHEK cells, endogenous NEDD8 interacted with EGFR through co-immunoprecipitation using NEDD8 antibody after IL-17 stimulation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). All data are expressed as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01 by one-way ANOVA with post hoc Tukey (A and B).

Article Snippet: For co-immunoprecipitation of UBE2M, NEDD8, or EGFR, protein A/G magnetic beads (MedChemExpress, Cat# HY-K0202) were pre-incubated with the indicated antibodies (5 μL per reaction) at 4°C for 6 h, followed by overnight incubation with lysates derived from one 10-cm culture dish per sample.

Techniques: Expressing, Control, Western Blot, Transfection, Lysis, Immunoprecipitation, Over Expression, Knockdown

Journal: iScience

Article Title: UBE2M-mediated EGFR neddylation drives keratinocyte proliferation in psoriasis

doi: 10.1016/j.isci.2026.115784

Figure Lengend Snippet: CUL4A-RBX1 and CUL4B-RBX1 act as E3 ligases for EGFR neddylation (A and B) The interaction of RBX1 and EGFR was detected by CoIP. HaCaT cells were transfected with Flag-EGFR and HA-RBX1 for 48 h, followed by anti-Flag (A) or anti-HA (B) immunoprecipitation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (C) The interaction of EGFR and NEDD8 with and without RBX1 knockdown using siRNA. Anti-Flag immunoprecipitation was employed as indicated. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (D) The neddylation of EGFR under various cullins knockdown conditions was detected by immunoprecipitation with a Flag antibody. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (E and F) The interaction of EGFR and CUL4A was detected by CoIP. HaCaT cells were transfected with Flag-EGFR and MYC-CUL4A for 48 h, followed by anti-Flag (E) or anti-MYC (F) immunoprecipitation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (G and H) The interaction of EGFR and CUL4B was detected by CoIP. HaCaT cells were transfected with Flag-EGFR and HA-CUL4B for 48 h, followed by anti-Flag (G) or anti-HA (H) immunoprecipitation. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (I and J) The physical interaction of NEDD8 and Flag-EGFR in HaCaT cells with and without CUL4A (I) or CUL4B (J) overexpression. Cell lysates were immunoprecipitated using Flag antibodies. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (K) The physical interaction of CUL4A and CUL4B with endogenous EGFR in HaCaT cells with and without UBE2M overexpression. Cell lysates were immunoprecipitated with IgG or EGFR antibodies. Representative immunoblots from one of three independent experiments are shown ( n = 3 independent repeat experiments). (L) A diagrammatic summary of how UBE2M promotes the progression of psoriasis by enhancing EGFR neddylation and stability.

Article Snippet: For co-immunoprecipitation of UBE2M, NEDD8, or EGFR, protein A/G magnetic beads (MedChemExpress, Cat# HY-K0202) were pre-incubated with the indicated antibodies (5 μL per reaction) at 4°C for 6 h, followed by overnight incubation with lysates derived from one 10-cm culture dish per sample.

Techniques: Transfection, Immunoprecipitation, Western Blot, Knockdown, Over Expression

Journal: Frontiers in Cellular Neuroscience

Article Title: Adipose-derived stem cell-conditioned medium mitigates ischemia-induced neuronal injury via the JAK1/STAT3 signaling pathway

doi: 10.3389/fncel.2026.1744887

Figure Lengend Snippet: ADSC-CM promotes neurite outgrowth in OGD-injured neurons through the JAK1-STAT3 signaling pathway. (A) On day 5 of cultivation, neuronal neurites extended and formed an extensive neural network. Immunofluorescence staining showed that the neurons were positive for the neuronal-specific marker Tuj1. After OGD injury, neuronal neurites were damaged, partially interrupted, and disappeared. (B) Western blot bands of JAK1, pJAK1, STAT3, pSTAT3, and GAPDH for each group. (C) Comparison of the relative ratio of pJAK1 to JAK1 across experimental groups ( n = 4). (D) Comparison of the relative ratio of pSTAT3 to STAT3 across experimental groups ( n = 4). (E) Immunofluorescence images of neurons from each group, showing the effects of ADSC-CM and GLPG0634 on neurite outgrowth. Neurons were stained with the Tuj1 antibody (red) to label the neuronal cytoskeleton, and nuclei were counterstained with DAPI (blue). (F) Diagram illustrating how to measure the length of the longest neurite and the number of primary neurites in neurons. The green line shows the trajectory of the longest neurite, and white arrows point to primary neurites. (G,H) Comparison of the length of the longest neurite and the number of primary neurites in neurons across experimental groups ( n = 4). Data are expressed as means ± SEM. The difference between the groups was assessed using a one-way ANOVA followed by Bonferroni post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 μm (A) , 20 μm (E,F) .

Article Snippet: After overnight incubation with rabbit anti-Tuj1 antibody (1:200, BM3881, Boster, Wuhan, China) at 4 °C, cells were washed and then incubated with Cy3-conjugated goat anti-rabbit IgG (1:400, AS007, ABclonal) for 1 h at room temperature in the dark.

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms

doi: 10.3389/fimmu.2018.01142

Figure Lengend Snippet: Confirmation of differentially expressed proteins and phosphoproteins by western blotting and Phos-tag western Blotting. (A) Analysis of ubiquitin conjugating enzyme E2 I, ubiquitin conjugating enzyme E2 L3, glyceraldehyde-3-phosphate dehydrogenase, β-actin expression levels in foot-and-mouth disease virus (FMDV)-infected and control cells by western blotting. SILAC-ratios and immunoblotting ratios (infection/control) were shown on the right side. (B) Analysis of the dynamic phosphorylation alterations of the three differentially phosphoproteins (ribosomal protein L15, chromosome 5 open reading frame 24, and FOS-like 2) in FMDV-infected and control cells by Phos-tag western blotting.

Article Snippet: To confirm the expression levels of GAPDH, ubiquitin conjugating enzyme E2 I (UBE2I), ubiquitin conjugating enzyme E2 L3 (UBE2L3), ribosomal protein L15 (RPL15), chromosome 5 open reading frame 24 (C5ORF24) and FOS-like 2 (FOSL2), anti-GAPDH antibody (Beyotime, China), anti-UBE2I antibody (Proteintech, China), anti-UBE2L3 antibody (Proteintech, China), anti-RPL15 antibody (Proteintech, China), anti-C5ORF24 antibody (Proteintech, China), and anti-FOSL2 antibody (Proteintech, China) were used for immunoblotting.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Virus, Infection, Control, Multiplex sample analysis, Phospho-proteomics