ethynyl  (Thermo Fisher)

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: EdU Cell Proliferation Kit , Med Chem Express , Cat#HY-K1087.

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 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: EdU Cell Proliferation Kit , Med Chem Express , Cat#HY-K1087.

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