Journal: Cells

Article Title: DCUN1D1 Is an Essential Regulator of Prostate Cancer Proliferation and Tumour Growth That Acts through Neddylation of Cullin 1, 3, 4A and 5 and Deregulation of Wnt/Catenin Pathway.

doi: 10.3390/cells12151973

Figure Lengend Snippet: Figure 5. Blockage of DCUN1D1 decreases global neddylation, ubiquitination and expression of neddylation components and shows preferential neddylation activity of cullin proteins in PCa. Western blot analysis of DU145 and DU145 DCUN1D1-KD cells. Protein extracts were obtained from the cells and subjected to Western blot analysis using (a) anti-ubiquitin, (b) anti-Nedd8. Blockage of DCUN1D1 decreased the expression of the neddylation pathway components including (c) the E1 NAE heterodimer APPB1 (top panel) and the neddylation-conjugating enzyme, UBC12 (bottom panel) and (d) the cullin-associated proteins RBX1 (top panel) and CAND1 (bottom panel). Inhibition of DCUN1D1 showed preferential NEDD8 modification of the cullin family of proteins. Immunoblot analysis of DU145 and DU145 DCUN1D1 knockdown cell protein extracts using (e) anti-cullin 1, (f) anti-cullin 3, (g) anti-cullin 4A, (h) anti-cullin 4B and (i) anti-cullin 5. The GAPDH loading control was probed using the anti-GAPDH antibody. Experiments were independently repeated three times and a representative image of an independent experiment is represented.

Article Snippet: The following primary antibodies were used: DCUN1D1 (Sigma HPA035911), Nedd8 (Cell Signaling Technology, Danver, MA, USA CST2745S), Ubiquitin (Cell Signaling Technology CST39365), cullin 1 (Cell Signaling Technology CST4995), cullin 2 (Novus Biologicals, Centennial, CO, USA NBP1-67535), cullin 3 (Cell Signaling Technology CST2759S), cullin 4A (Cell Signaling Technology CST2699S), cullin 4B (Bio-Rad VMA00360) Cells 2023, 12, 1973 6 of 22 cullin 5 (Novus Biologicals NBP1-22970), APPBP1 (Cell Signaling Technology CST14321), UBA3 (Novus Biologicals NBP2-48628), UBC12 (Novus Biologicals NBP1-31459), CAND1 (Cell Signaling Technology CST8759S), RBX1 (Novus Biologicals NBP2-20113), β-Catenin (Cell Signaling Technology CST4394S) phosphor-β-Catenin (Cell Signaling Technology CST2009S) and Lef1 (Santa Cruz sc-374522).

Techniques: Ubiquitin Proteomics, Expressing, Activity Assay, Western Blot, Inhibition, Knockdown, Control

Journal: The EMBO Journal

Article Title: RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis

doi: 10.1038/s44318-024-00353-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Anti-UBE2M antibody , Santa Cruz , Cat #sc-100608; RRID: AB_2211030.

Techniques: Mutagenesis, Recombinant, CCK-8 Assay, Software

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability

doi: 10.1096/fj.202001146RR

Figure Lengend Snippet: Inhibition of neddylation stabilizes antithrombin. A,B, Assessment of antithrombin expression in HepG2 and SMMC7721 cells after exposure to the indicated concentrations of MLN4924 for 24 hours or to 1 µM MLN4924 for the indicated times. A, Representative western blots showing antithrombin protein expression. B, Statistical analysis of antithrombin protein expression. C, The half-life of antithrombin was evaluated by exposing HepG2 and SMMC7721 cells to CHX alone or in combination with MLN4924 for the indicated times. D, HepG2 and SMMC7721 cells were treated with MLN4924 for 24 hours, followed by treatment with 100 nM bortezomib for an additional 2 hours. Total polyubiquitinated antithrombin was evaluated by immunoprecipitation. E-G, Western blot analysis of the neddylation proteins, UBA3 (E), NAE1 (F), and UBE2M (G), in HepG2 and SMMC7721 cells 48 hours after transfection with 50 nM siRNAs against the corresponding enzymes

Article Snippet: Medium was replaced with fresh medium, as appropriate, after transfection for 6 hours and cells were cultured for 48 or 72 hours for further analysis. siRNAs against USP2 (sc-76821), UBA3 (sc-76783), NAE1 (sc-72523), and UBE2M (sc-76786) were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Expressing, Western Blot, Immunoprecipitation, Transfection

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: Experimental & Molecular Medicine

Article Title: Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals

doi: 10.1038/s12276-021-00585-z

Figure Lengend Snippet: a HEK 293 T cells were cotransfected with myc-mGlu7 and HA-NEDD8 or HA-NEDD8ΔGG and treated with 1 μM MLN4924 or vehicle (DMSO) for 6 h. Thirty-six hours after transfection, cell lysates were immunoprecipitated with an anti-myc antibody. Western blotting was performed with anti-HA or anti-myc antibodies. *, nonspecific bands; MLN, MLN4924. b HEK 293 T cells were cotransfected with myc-mGlu7, HA-NEDD8, and UBC12 C111S-His or UBC12 WT-His. Immunoprecipitation and western blotting were performed as shown in panel a . An open triangle indicates the UBC12 C111S-NEDD8 complex. *, nonspecific bands. c Neddylation of endogenous mGlu7 is blocked by MLN4924. Primary cortical neurons (DIV14) were treated with 1 μM MLN4924 or vehicle for 6 h. Cell lysates were immunoprecipitated with anti-mGlu7 antibody and subjected to western blotting using the indicated antibodies. d , e Agonist stimulation reduces mGlu7 neddylation. Primary cortical neurons (DIV14) were treated with 400 μM L-AP4 for 5 min at 37 °C. Cell lysates were immunoprecipitated with an anti-mGlu7 antibody ( d ) or an anti-NEDD8 antibody ( e ). The immunoprecipitates were analyzed by western blotting. The bar graph with scatter plots shows the mean ± SEM (mGlu7 HMW, vehicle: 1.00 ± 0.06, L-AP4: 0.59 ± 0.01; mGlu7 LMW, vehicle: 1.00 ± 0.01, L-AP4: 0.32 ± 0.06; n = 3, * p < 0.05, Student’s t test).

Article Snippet: UBC12 C111S-His and NEDD8ΔGG were generated from UBC12 wild-type (WT)-His (# 20080, Addgene) and NEDD8, respectively, by site-directed mutagenesis.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Neddylation is required for presynaptic clustering of mGlu7 and maturation of presynaptic terminals

doi: 10.1038/s12276-021-00585-z

Figure Lengend Snippet: a Primary hippocampal neurons (DIV10) were cotransfected with myc-mGlu7 and control vector (vec), UBC12 C111S, or UBC12 WT. Four days after transfection, the surface-expressed mGlu7 (red) and the internalized mGlu7 (green) were analyzed from Z-stacked maximal projection images using confocal microscopy. The representative images are presented. Scale bar, 20 μm. The summary quantification graph indicates the amount of internalized versus total (surface + internalized) mGlu7 using Metamorph software. The bar graph with scatter plots shows the mean ± SEM (vec, 1.00 ± 0.02; UBC12 C111S, 0.98 ± 0.03; UBC12 WT, 1.10 ± 0.05; vec + L-AP4, 1.22 ± 0.03; UBC12 C111S + L-AP4, 1.20 ± 0.02; UBC12 WT + L-AP4, 1.30 ± 0.04; n = 3, total neurons > 20; * p < 0.05, n.s. indicates p > 0.05, one-way ANOVA followed by Tukey’s post hoc test). b , c Surface biotinylation assay in cultured cortical neurons (DIV14). Neurons were pretreated with 1 μM MLN4924 or vehicle for 6 h and then unstimulated ( b ) or stimulated ( c ) with 400 μM L-AP4 for 15 min. Surface-expressed endogenous mGlu7 was biotinylated with membrane-impermeable Sulfo-NHS-SS-Biotin. After washing, the lysates were pulled down using streptavidin-agarose beads. The bead-bound proteins were detected by western blotting using an anti-mGlu7 antibody. The bar graph with scatter plots shows the mean ± SEM ( b , DMSO, 1.00 ± 0.10; MLN, 1.07 ± 0.05; n = 6; n.s. indicates p > 0.05, Student’s t test. c , DMSO, 1.00 ± 0.11; MLN, 0.85 ± 0.08; n = 5; n.s. indicates p > 0.05, Student’s t test). To measure the effect of MLN4924, the bead-bound surface fraction was eluted using 50 mM DTT for 30 min at 50 °C. The eluted surface fraction was then immunoprecipitated using an anti-mGlu7 antibody and blotted with an anti-NEDD8 or anti-mGlu7 antibody.

Article Snippet: UBC12 C111S-His and NEDD8ΔGG were generated from UBC12 wild-type (WT)-His (# 20080, Addgene) and NEDD8, respectively, by site-directed mutagenesis.

Techniques: Control, Plasmid Preparation, Transfection, Confocal Microscopy, Software, Surface Biotinylation Assay, Cell Culture, Membrane, Western Blot, Immunoprecipitation