p mkk7  (Proteintech)

Journal: Journal of Translational Medicine

Article Title: UBE2M inhibits neoplastic cell proliferation via MKK7–JNK–EGR1 axis in melanoma

doi: 10.1186/s12967-025-07531-x

Figure Lengend Snippet: UBE2M enhances EGR1 expression via activation of the MKK7-JNK pathway. ( A ) and ( B ) The physical interaction of NEDD8 and Ub with EGR1-Flag in 293 T cells upon UBE2M overexpression. Cell lysates were immunoprecipitated using Flag antibodies, alongside quantification analysis. ( C ) Western blot analysis of total and phosphorylated JNK, ERK, and p38 in melanoma cells with UBE2M overexpression, alongside statistical analysis of the indicated proteins. ( D ) The transcription of JNK in melanoma cells with UBE2M overexpression examined by RT-qPCR. ( E ) JNK inhibitor JNK-IN-8 blocked the UBE2M-mediated upregulation of EGR1 in A375 cell lines. The cells were administered JNK-IN-8 (1 µM, 48 h) before harvesting. Quantification was employed as indicated. ( F ) Western blot analysis of total and phosphorylated MKK7 and MKK4 in melanoma cells with UBE2M overexpression, alongside statistical analysis of the indicated proteins. ( G ) The transcription of MKK7 in melanoma cells with UBE2M overexpression examined by RT-qPCR. ( H ) MKK7 knockdown blocked the UBE2M-mediated upregulation of JNK phosphorylation and EGR1 expression in A375 cell lines. The cells were transfected with plasmids encoding UBE2M and siRNA targeting MKK7 before harvesting. The data are presented as the means ± SD. ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, according to unpaired Student’s t – test ( A - D , F , G ) and one-way ANOVA with post hoc Tukey ( E , H ). n = 3

Article Snippet: The antibodies we used were as follows: UBE2M (Proteintech, #14520-1-AP, 1:1000), GAPDH (Proteintech, #60004-1-Ig, 1:5000), NEDD8 (Cell Signaling Technology, #2745, 1:1000), EGR1 (ABclonal, #A7266, 1:600), HA (Proteintech, #51064-2-AP, 1:7000), Flag (Proteintech, #20543-1-AP, 1:20000), Ubiquitin (Proteintech, #10201-2-AP, 1:2000), JNK (Proteintech, #51153-1-AP, 1:1000), p-JNK (Proteintech, #80024-1-RR, 1:2000), ERK1/2 (Proteintech, #11257-1-AP, 1:3000), p-ERK1/2 (Cell Signaling Technology, #4370, 1:2000), p38 (Proteintech, #14064-1-AP, 1:5000), p-p38 (Proteintech, #28796-1-AP, 1:2000), MKK7 (Proteintech, #55030-1-AP, 1:500), p-MKK7 (Proteintech, #80357-1-RR, 1:5000), MKK4 (Proteintech, #17340-1-AP, 1:1000), p-MKK4 (Proteintech, #29250-1-AP, 1:1000), UBE2F (Proteintech, #17056-1-AP, 1:1000), CCND2 (Proteintech, #10934-1-AP, 1:1000), CCND1 (Proteintech, #26939-1-AP, 1:20000), CDK4 (Proteintech, #11026-1-AP, 1:4000), CDK6 (Proteintech, #14052-1-AP, 1:3000), goat anti-mouse IgG (Proteintech, #SA00001-1), and goat anti-rabbit IgG (Proteintech, #SA00001-2).

Techniques: Expressing, Activation Assay, Over Expression, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Knockdown, Phospho-proteomics, Transfection

Journal: Journal of Translational Medicine

Article Title: UBE2M inhibits neoplastic cell proliferation via MKK7–JNK–EGR1 axis in melanoma

doi: 10.1186/s12967-025-07531-x

Figure Lengend Snippet: UBE2M is the E2 enzyme for MKK7 neddylation. ( A ) UBE2M-Flag was transfected into 293 T cells and treated with 0.3 µM MLN4924 for 12 h. Cell lysis was followed by IP and quantification analysis. ( B , C ) MKK7-Flag or UBE2M was co-transfected with different tagged forms of NEDD8 into 293T cells. 0.3 µM MLN4924 was employed for 12 h. Cell lysis was followed by anti-Flag IP and quantification analysis as indicated. ( D ) The interaction of MKK7 and NEDD8 in 293 T cells with overexpression of UBE2M or UBE2F. Anti-Flag IP and quantification analysis were employed as indicated. *Denotes the band detected under co-incubation with UBE2M and UBE2F antibodies. The data are presented as the means ± SD. * P < 0.05; ** P < 0.01, *** P < 0.001, according to unpaired Student’s t – test ( A , B ) and one-way ANOVA with post hoc Tukey ( C , D ). n = 3

Article Snippet: The antibodies we used were as follows: UBE2M (Proteintech, #14520-1-AP, 1:1000), GAPDH (Proteintech, #60004-1-Ig, 1:5000), NEDD8 (Cell Signaling Technology, #2745, 1:1000), EGR1 (ABclonal, #A7266, 1:600), HA (Proteintech, #51064-2-AP, 1:7000), Flag (Proteintech, #20543-1-AP, 1:20000), Ubiquitin (Proteintech, #10201-2-AP, 1:2000), JNK (Proteintech, #51153-1-AP, 1:1000), p-JNK (Proteintech, #80024-1-RR, 1:2000), ERK1/2 (Proteintech, #11257-1-AP, 1:3000), p-ERK1/2 (Cell Signaling Technology, #4370, 1:2000), p38 (Proteintech, #14064-1-AP, 1:5000), p-p38 (Proteintech, #28796-1-AP, 1:2000), MKK7 (Proteintech, #55030-1-AP, 1:500), p-MKK7 (Proteintech, #80357-1-RR, 1:5000), MKK4 (Proteintech, #17340-1-AP, 1:1000), p-MKK4 (Proteintech, #29250-1-AP, 1:1000), UBE2F (Proteintech, #17056-1-AP, 1:1000), CCND2 (Proteintech, #10934-1-AP, 1:1000), CCND1 (Proteintech, #26939-1-AP, 1:20000), CDK4 (Proteintech, #11026-1-AP, 1:4000), CDK6 (Proteintech, #14052-1-AP, 1:3000), goat anti-mouse IgG (Proteintech, #SA00001-1), and goat anti-rabbit IgG (Proteintech, #SA00001-2).

Techniques: Transfection, Lysis, Over Expression, Incubation

Journal: Journal of Translational Medicine

Article Title: UBE2M inhibits neoplastic cell proliferation via MKK7–JNK–EGR1 axis in melanoma

doi: 10.1186/s12967-025-07531-x

Figure Lengend Snippet: Neddylation stabilizes MKK7 by preventing its degradation via the ubiquitin-proteasome pathway. ( A ) The changes of the MKK7 protein level in A375 cells with or without ectopic UBE2M overexpression under the treatment of CHX (50 µg/mL). MKK7 levels were analyzed by western blots, and the relative density of protein bands were quantified. n = 3. ( B ) The changes of MKK7 levels were analyzed in A375 cells with MG132 (20 µM, 10 h) or CQ (50 µM, 10 h) treatment. n = 3. ( C ) The Ub modification of MKK7 was reduced upon UBE2M overexpression and was increased upon MLN4924 treatment in A375 cells detected by IP. The relative quantification of Ub modification was performed. n = 3. ( D ) PLA analysis of the interaction between MKK7 and Ub. A375 cells were transfected with UBE2M and negative control. PLA was conducted with antibodies against MKK7 and Ub. Nuclei were stained with DAPI (blue). PLA puncta (red) for each condition were imaged. (Scale bar, 10 μm.) The quantification of PLA was plotted. n = 19. ( E ) Western blot analysis of cell cycle proteins in A375 cells with UBE2M overexpression, alongside statistical analysis of the indicated proteins. n = 3. ( F ) EGR1 knockdown blocked the UBE2M-mediated downregulation of CCND2 in A375 cell lines. The cells were transfected with plasmids encoding UBE2M and siRNA targeting EGR1 before harvesting. n = 3. ( G , H ) CCK-8 assay analysis of cell proliferation of A375 and M14 cells upon UBE2M overexpression with or without EGR1 knockdown. The NC groups used were A375 and M14 cells stably transfected with an empty vector. Quantification was employed as indicated ( n = 3). The data are presented as the means ± SD. ns, not significant; * P < 0.05; ** P < 0.01, *** P < 0.001; **** P < 0.0001, according to two-way ANOVA with post hoc Tukey ( A , G , H ), unpaired Student’s t – test ( B , D , E ) and one-way ANOVA with post hoc Tukey ( C , F )

Article Snippet: The antibodies we used were as follows: UBE2M (Proteintech, #14520-1-AP, 1:1000), GAPDH (Proteintech, #60004-1-Ig, 1:5000), NEDD8 (Cell Signaling Technology, #2745, 1:1000), EGR1 (ABclonal, #A7266, 1:600), HA (Proteintech, #51064-2-AP, 1:7000), Flag (Proteintech, #20543-1-AP, 1:20000), Ubiquitin (Proteintech, #10201-2-AP, 1:2000), JNK (Proteintech, #51153-1-AP, 1:1000), p-JNK (Proteintech, #80024-1-RR, 1:2000), ERK1/2 (Proteintech, #11257-1-AP, 1:3000), p-ERK1/2 (Cell Signaling Technology, #4370, 1:2000), p38 (Proteintech, #14064-1-AP, 1:5000), p-p38 (Proteintech, #28796-1-AP, 1:2000), MKK7 (Proteintech, #55030-1-AP, 1:500), p-MKK7 (Proteintech, #80357-1-RR, 1:5000), MKK4 (Proteintech, #17340-1-AP, 1:1000), p-MKK4 (Proteintech, #29250-1-AP, 1:1000), UBE2F (Proteintech, #17056-1-AP, 1:1000), CCND2 (Proteintech, #10934-1-AP, 1:1000), CCND1 (Proteintech, #26939-1-AP, 1:20000), CDK4 (Proteintech, #11026-1-AP, 1:4000), CDK6 (Proteintech, #14052-1-AP, 1:3000), goat anti-mouse IgG (Proteintech, #SA00001-1), and goat anti-rabbit IgG (Proteintech, #SA00001-2).

Techniques: Ubiquitin Proteomics, Over Expression, Western Blot, Modification, Quantitative Proteomics, Transfection, Negative Control, Staining, Knockdown, CCK-8 Assay, Stable Transfection, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: UBE2M inhibits neoplastic cell proliferation via MKK7–JNK–EGR1 axis in melanoma

doi: 10.1186/s12967-025-07531-x

Figure Lengend Snippet: UBE2M-mediated neddylation of MKK7 upregulates EGR1 to inhibit melanoma proliferation. UBE2M mediates MKK7 neddylation in melanoma cells, reducing its ubiquitination and proteasomal degradation, thereby increasing MKK7 protein levels. Elevated MKK7 phosphorylation promotes JNK activation in the MAPK pathway, leading to enhanced transcription and expression of EGR1. EGR1 subsequently represses CCND2 expression, resulting in inhibited melanoma cell proliferation

Article Snippet: The antibodies we used were as follows: UBE2M (Proteintech, #14520-1-AP, 1:1000), GAPDH (Proteintech, #60004-1-Ig, 1:5000), NEDD8 (Cell Signaling Technology, #2745, 1:1000), EGR1 (ABclonal, #A7266, 1:600), HA (Proteintech, #51064-2-AP, 1:7000), Flag (Proteintech, #20543-1-AP, 1:20000), Ubiquitin (Proteintech, #10201-2-AP, 1:2000), JNK (Proteintech, #51153-1-AP, 1:1000), p-JNK (Proteintech, #80024-1-RR, 1:2000), ERK1/2 (Proteintech, #11257-1-AP, 1:3000), p-ERK1/2 (Cell Signaling Technology, #4370, 1:2000), p38 (Proteintech, #14064-1-AP, 1:5000), p-p38 (Proteintech, #28796-1-AP, 1:2000), MKK7 (Proteintech, #55030-1-AP, 1:500), p-MKK7 (Proteintech, #80357-1-RR, 1:5000), MKK4 (Proteintech, #17340-1-AP, 1:1000), p-MKK4 (Proteintech, #29250-1-AP, 1:1000), UBE2F (Proteintech, #17056-1-AP, 1:1000), CCND2 (Proteintech, #10934-1-AP, 1:1000), CCND1 (Proteintech, #26939-1-AP, 1:20000), CDK4 (Proteintech, #11026-1-AP, 1:4000), CDK6 (Proteintech, #14052-1-AP, 1:3000), goat anti-mouse IgG (Proteintech, #SA00001-1), and goat anti-rabbit IgG (Proteintech, #SA00001-2).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Activation Assay, Expressing

Journal: Chinese Herbal Medicines

Article Title: Ganoderic acid a derivative induces apoptosis of cervical cancer cells by inhibiting JNK pathway

doi: 10.1016/j.chmed.2024.07.002

Figure Lengend Snippet: (A) Target prediction analysis results. (B) Prediction of GaAD19-JNK interaction. (C–E) Western blot analysis of HeLa cells with GaAD19 treatment for 24 h on expression of JNK, p-JNK, ERK, p-ERK, p38, p-p38 (C), AMPK α 1, p-AMPK α 1-S485, p-AMPK α 1-S496, AMPK β 1, p-AMPK β 1-S108, Akt, p-Akt (D), MAP2K4, MAP2K7 (E), and other proteins (means ± SD, n = 3). (F) Effect of GaAD19 on mRNA level of JNK pathway membrane protein receptor in HeLa cells was determined by qPCR (means ± SD, n = 3). ( ** P < 0.01, *** P < 0.001 vs control group, ns indicates no significant difference).

Article Snippet: MAP2K7 , Proteintech , 55030–1-AP , 1:500.

Techniques: Western Blot, Expressing, Membrane, Control