anti mxi1  (Proteintech)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: FUBP3 mediates MXI1 stability to silence RRAS and hinder MAPK signaling in acute megakaryoblastic leukemia progression

doi: 10.1007/s00262-025-04257-z

Figure Lengend Snippet: MXI1 represses the transcription of RRAS by recruiting NCOR1/2, Sin3A/B, and HDAC1. ( A ) Detection of MXI1 mRNA expression in PBMCs, monocytes, and AMKL cells by RT-qPCR (n = 5, one-way ANOVA). ( B ) Detection of MXI1 protein expression in PBMCs, monocytes, and AMKL cells by western blot analysis (n = 3, one-way ANOVA). ( C ) MXI1 expression in AMKL cells by RT-qPCR assay (n = 5, unpaired t-test) and western blot analysis (n = 3, unpaired t-test). ( D ) RRAS expression in AMKL cells by RT-qPCR assay (n = 5, unpaired t-test). ( E ) Enrichment of the RRAS promoter fragments by anti-MXI1, anti-NCOR1/2, anti-Sin3A/B, and anti-HDAC1 in AMKL cells using ChIP assay (n = 5, one-way ANOVA). ( F ) Interaction between MXI1 and RRAS promoters in AMKL cells was verified using a dual-luciferase reporter gene assay (n = 5, unpaired t-test). ( G ) The phosphorylation and total protein of ERK-1/2 in AMKL cells overexpressing MXI1 were determined by western blot assay (n = 3, unpaired t-test). Data were presented as means ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The transfer membranes were blocked in 5% non-fat milk or 5% BSA at RT for 1 h and incubated with the primary antibodies to RRAS (1/1000, PA5-141,028, Thermo Fisher), Phospho (p)-ERK-1/2 (1/1000, 44-680G, Thermo Fisher), ERK-1/2 (1/1000, A4782, Abclonal), PD-L1 (1/1000, MA5-38,419, Thermo Fisher), MXI1 (1/500, 12,360–1-AP, ProteinTech Group), FUBP3 (1/1000, MBS9434216, MyBioSource), Cleaved caspase-3 (1/100, ab2302, Abcam), and GAPDH (1/2500, ab9485, Abcam) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Gene Assay, Phospho-proteomics

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: FUBP3 mediates MXI1 stability to silence RRAS and hinder MAPK signaling in acute megakaryoblastic leukemia progression

doi: 10.1007/s00262-025-04257-z

Figure Lengend Snippet: RRAS activates MAPK signaling and suppresses CD8 + T cell immunoreactivity in the presence of MXI1 . ( A ) Event-free survival of AML patients with different MXI1 expression analyzed by Kaplan–Meier Plotter. ( B ) Detection of RRAS expression in AMKL cells after infection of oe- MXI1 + oe-NC or oe- RRAS by RT-qPCR (n = 5, unpaired t-test) and western blot analysis (n = 3, unpaired t-test). ( C ) The extent of ERK-1/2 phosphorylation in AMKL cells was determined by western blot assay (n = 3, unpaired t-test). ( D ) The proliferative capacity of CD8 + T cells was analyzed by flow cytometry (n = 5, one-way ANOVA). ( E ) CD8 + T cell surface activation marker CD25 expression and cytotoxic molecule GzmB production were analyzed by flow cytometry (n = 5, one-way ANOVA). ( F ) Levels of IL-2 and IFN-γ in co-culture systems were detected by ELISA (n = 5, one-way ANOVA). ( G ) The proportion of PD-1 + CD8 + and TIM-3 + CD8 + in a co-culture system was analyzed by flow cytometry (n = 5, one-way ANOVA). ( H ) Protein expression of PD-L1 on the surface of AMKL cells was analyzed using western blot analysis (n = 3, one-way ANOVA). (I) PD-L1 expression on the surface of AMKL cells was analyzed using flow cytometry (n = 5, one-way ANOVA). ( J ) AMKL cell viability detected by CCK-8 (n = 5, one-way ANOVA). ( K ) AMKL cell apoptosis detected by TUNEL assay. Data were presented as means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The transfer membranes were blocked in 5% non-fat milk or 5% BSA at RT for 1 h and incubated with the primary antibodies to RRAS (1/1000, PA5-141,028, Thermo Fisher), Phospho (p)-ERK-1/2 (1/1000, 44-680G, Thermo Fisher), ERK-1/2 (1/1000, A4782, Abclonal), PD-L1 (1/1000, MA5-38,419, Thermo Fisher), MXI1 (1/500, 12,360–1-AP, ProteinTech Group), FUBP3 (1/1000, MBS9434216, MyBioSource), Cleaved caspase-3 (1/100, ab2302, Abcam), and GAPDH (1/2500, ab9485, Abcam) at 4 °C overnight.

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Flow Cytometry, Activation Assay, Marker, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, TUNEL Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: FUBP3 mediates MXI1 stability to silence RRAS and hinder MAPK signaling in acute megakaryoblastic leukemia progression

doi: 10.1007/s00262-025-04257-z

Figure Lengend Snippet: FUBP3 induces MXI1 degradation in AMKL. ( A ) Correlation analysis of FUBP3 and MXI1 expression in LAML tumor by GEPIA correlation. ( B ) FUBP3 expression in PBMCs, monocytes, and AMKL cells by RT-qPCR (n = 5, one-way ANOVA). ( C ) FUBP3 protein expression in PBMCs, monocytes, and AMKL cells by western blot analysis (n = 3, one-way ANOVA). ( D ) FUBP3 protein pulled down by biotin-labeled MXI1 in AMKL cell lysates was examined using RNA pull-down assay (n = 3). ( E ) Overexpression efficiency of FUBP3 in AMKL cells was detected by RT-qPCR (n = 5, unpaired t-test). ( F ) Regulation of MXI1 by anti-FUBP3 in AMKL cells after overexpression of FUBP3 detected by RIP-qPCR (n = 5, two-way ANOVA). ( G ) The effect of overexpression of FUBP3 on MXI1 mRNA stability at different time points after treatment of AMKL cells with 5 μg/mL actinomycin D was analyzed by RT-qPCR (n = 5, two-way ANOVA). Data were presented as means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The transfer membranes were blocked in 5% non-fat milk or 5% BSA at RT for 1 h and incubated with the primary antibodies to RRAS (1/1000, PA5-141,028, Thermo Fisher), Phospho (p)-ERK-1/2 (1/1000, 44-680G, Thermo Fisher), ERK-1/2 (1/1000, A4782, Abclonal), PD-L1 (1/1000, MA5-38,419, Thermo Fisher), MXI1 (1/500, 12,360–1-AP, ProteinTech Group), FUBP3 (1/1000, MBS9434216, MyBioSource), Cleaved caspase-3 (1/100, ab2302, Abcam), and GAPDH (1/2500, ab9485, Abcam) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Labeling, Pull Down Assay, Over Expression

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: FUBP3 mediates MXI1 stability to silence RRAS and hinder MAPK signaling in acute megakaryoblastic leukemia progression

doi: 10.1007/s00262-025-04257-z

Figure Lengend Snippet: FUBP3 mediates MXI1 stability by recruiting co-inhibitors to silence RRAS . ( A ) MXI1 mRNA expression in AMKL cells after infection of oe- FUBP3 + sh-NC or sh- MXI1 1#, 2#, and 3# by RT-qPCR (n = 5, one-way ANOVA). ( B ) MXI1, RRAS, and ERK-1/2 protein expression and the extent of ERK-1/2 phosphorylation in AMKL cells after infection were analyzed by western blot assay (n = 3, one-way ANOVA). ( C ) The proliferative capacity of CD8 + T cells was analyzed by flow cytometry (n = 5, one-way ANOVA). ( D ) CD8 + T cell surface activation marker CD25 expression and cytotoxic molecule GzmB production were analyzed by flow cytometry (n = 5, one-way ANOVA). ( E ) Levels of IL-2 and IFN-γ in co-culture systems were detected by ELISA (n = 5, one-way ANOVA). ( F ) The proportion of PD-1 + CD8 + and TIM-3 + CD8 + in a co-culture system was analyzed by flow cytometry (n = 5, one-way ANOVA). ( G ) Protein expression of PD-L1 on the surface of AMKL cells was analyzed using western blot analysis (n = 3, one-way ANOVA). ( H ) PD-L1 expression on the surface of AMKL cells was analyzed using flow cytometry (n = 5, one-way ANOVA). ( I ) AMKL cell viability detected by CCK-8 (n = 5, one-way ANOVA). ( J ) AMKL cell apoptosis detected by TUNEL assay (n = 5, one-way ANOVA). ( K ) Protein expression of Cleaved caspase-3 on the surface of AMKL cells was analyzed using western blot analysis (n = 3, one-way ANOVA). Data were presented as means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The transfer membranes were blocked in 5% non-fat milk or 5% BSA at RT for 1 h and incubated with the primary antibodies to RRAS (1/1000, PA5-141,028, Thermo Fisher), Phospho (p)-ERK-1/2 (1/1000, 44-680G, Thermo Fisher), ERK-1/2 (1/1000, A4782, Abclonal), PD-L1 (1/1000, MA5-38,419, Thermo Fisher), MXI1 (1/500, 12,360–1-AP, ProteinTech Group), FUBP3 (1/1000, MBS9434216, MyBioSource), Cleaved caspase-3 (1/100, ab2302, Abcam), and GAPDH (1/2500, ab9485, Abcam) at 4 °C overnight.

Techniques: Expressing, Infection, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Flow Cytometry, Activation Assay, Marker, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, TUNEL Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: FUBP3 mediates MXI1 stability to silence RRAS and hinder MAPK signaling in acute megakaryoblastic leukemia progression

doi: 10.1007/s00262-025-04257-z

Figure Lengend Snippet: Knockdown of MXI1 upregulates RRAS and activates the MAPK signaling to suppress the anti-tumor immunity of CD8 + T cells. ( A ) WBC counts in the peripheral blood of NOG mice injected with M-07e cells infected with oe-NC, oe- FUBP3 , oe- FUBP3 + sh-NC, or oe- FUBP3 + sh- MXI1 #2 (n = 5, one-way ANOVA). ( B ) The spleen and liver tissues of mice injected with M-07e cells (n = 5, one-way ANOVA). ( C ) The positive rate of FUBP3, MXI1, and RRAS in the spleen tissues of AMKL mice was detected by immunohistochemistry (n = 5, one-way ANOVA). ( D ) The phosphorylation and total protein of ERK-1/2 in the spleen tissues of AMKL mice were detected by western blot analysis (n = 3, one-way ANOVA). ( E ) Representative HE images of bone marrow, liver, and spleen sections of mice (n = 5, one-way ANOVA). ( F ) Detection of CD41 and CD8 in single-cell suspensions from mouse spleens by flow cytometry (n = 5, one-way ANOVA). ( G ) Detection of CD8 in the spleen tissues of mice by immunofluorescence staining (n = 5, one-way ANOVA). ( H ) Detection of IFN-γ + CD8 + in single-cell suspensions from mouse spleens by flow cytometry (n = 5, one-way ANOVA). ( I ) Expression of GzmB + CD8 + and PRF1 + CD8 + in single-cell suspensions of mouse spleen detected by flow cytometry (n = 5, one-way ANOVA). Data were presented as means ± SEM, one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The transfer membranes were blocked in 5% non-fat milk or 5% BSA at RT for 1 h and incubated with the primary antibodies to RRAS (1/1000, PA5-141,028, Thermo Fisher), Phospho (p)-ERK-1/2 (1/1000, 44-680G, Thermo Fisher), ERK-1/2 (1/1000, A4782, Abclonal), PD-L1 (1/1000, MA5-38,419, Thermo Fisher), MXI1 (1/500, 12,360–1-AP, ProteinTech Group), FUBP3 (1/1000, MBS9434216, MyBioSource), Cleaved caspase-3 (1/100, ab2302, Abcam), and GAPDH (1/2500, ab9485, Abcam) at 4 °C overnight.

Techniques: Knockdown, Injection, Infection, Immunohistochemistry, Phospho-proteomics, Western Blot, Flow Cytometry, Immunofluorescence, Staining, Expressing