antibodies against sumo 1  (Developmental Studies Hybridoma Bank)

Journal: Frontiers in Immunology

Article Title: RMRP accelerates ligamentum flavum hypertrophy by regulating GSDMD-mediated pyroptosis through Gli1 SUMOylation

doi: 10.3389/fimmu.2024.1427970

Figure Lengend Snippet: RMRP stimulate fibrogenesis in vitro through the activation of Hedgehog-Gli1 signaling. (A) GO analysis for dysregulated genes in LF cells. (B) Western blot analysis of Gli1 and RMRP association in RNA pull-down assay. (C) RIP assay using Gli1 antibody, detection of RMRP or GAPDH by specific primers, and quantification of RIP enrichment (D, E) Increased RMRP expression enhanced its interaction with Gli1, as shown by RIP assay with Gli1 antibody. (n=3). (F, G) Co-IP (F) and reverse Co-IP (G) were used to assess interaction of Gli1 with SUMO1 examined by immunoblotting with antibodies (n=3). (H) HEK 293T cell lysate transfected with Myc-Gli1 and Flag-SUMO1 was immunoprecipitated with anti-Flag and immunoblotted with anti-Flag and anti-Myc (n=3). (I) Lysates from LF cells transfected with LV-control or LV-RMRP were immunoprecipitated and analyzed with specific antibodies to study the impact of RMRP on Gli1 SUMOylation (n=3). (J) RMRP’s influence on Gli1 translocation to the nucleus in LF cells, measured with immunofluorescence. Scale bar, 100μm. (K) Examining the co-localization of Gli1 and SUMO1 proteins using immunofluorescence analysis. Scale bar, 20μm. (L, M) Western blot analysis of the impact of Hedgehog pathway on RMRP-induced collagen expression. # P < 0.05, ## P < 0.01. LF, ligamentum flavum.

Article Snippet: The membranes were blocked in Blocking Buffer (Sigma) and then incubated at 4°C overnight with primary antibodies against SUMO1 (1:1000, ab32058, Abcam), ASC (1:1000, ab283684, Abcam), Gli1 (1:1000, ab167388, Abcam), Caspase-1 (1:500, ab238972, Abcam), Cleaved Caspase-1 (1:1000, bs-1247R, Bioss), GSDMD-N (1:500, ab215203, Abcam); GSDMD (1:1000, ab219800, Abcam), collagen I (1:1000, 14695-1-AP, Proteintech), collagen III (1:1000, bs-0549R, Bioss), NLRP3 (1:500, ab263899, Abcam), GAPDH (1:1000, AP0063, Bioworld), and α-SMA (1:1000, 55135-1-AP, Proteintech).

Techniques: In Vitro, Activation Assay, Western Blot, Pull Down Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Control, Translocation Assay, Immunofluorescence

Journal: bioRxiv

Article Title: The SUMOylation inhibitor TAK-981 (Subasumstat) triggers IFN-I-dependent activation of Natural Killer cells against Acute Myeloid Leukemias

doi: 10.1101/2024.02.19.580882

Figure Lengend Snippet: A. Experimental procedure: primary NK cells were purified from healthy donors’ PBMCs. NK cells were treated with TAK-981 for 24 h and co-cultured or not with AML cells. When co-cultured, NK cells activation, cytotoxicity and AML survival were measured after 4 h of co-culture. B. Immunoblot with SUMO-1 and SUMO-2/3 antibodies of purified NK cells treated with 100 nM TAK-981 for the indicated times. Amido-black staining was used as loading control. C. NK cells (upper panel) or total PBMCs (lower panel) collected from (n=5) healthy donors were treated with increasing concentrations of TAK-981 for 24 h. Cell viability was determined by flow cytometry and a dose-response curve was generated by comparing the viability of TAK-981 treated cells with mock-treated controls. Data are shown as mean +/− SEM of 5 donors. D. Violin plot showing the expression of CD69 on the surface of purified NK cells. NK cells were treated with 100 nM TAK-981 for 24 h, followed by 4 h of co-culture with U937 (Effector:Target (E:T) ratio 1:1). Median fluorescent intensity of CD69 was normalized to mock-treated condition without co-culture (n= 10 donors, RM one-way ANOVA test). E. Fox chase SCID mice were injected intravenously with 15 mg/kg TAK-981 or vehicle. Mice were euthanized after 5h, 24h or 48h and intraperitoneal cavity wash was performed to collect NK cells. F. Gating strategies for mouse NK cells: total cells purified from the intraperitoneal cavity were stained with CD3 and NKp46 antibodies. NK cells were gated as SSC low CD3-NKp46+. G. CD69 expression levels on NK cells from SCID mice treated or not with TAK-981 for indicated time (4 or 5 mice per group, RM one-way ANOVA).

Article Snippet: Antibodies against SUMO-1 (21C7), and SUMO2/3 (8A2) were obtained from the Developmental Studies Hybridoma Bank.

Techniques: Purification, Cell Culture, Activation Assay, Co-Culture Assay, Western Blot, Staining, Flow Cytometry, Generated, Expressing, Injection

Journal: Cell Death & Disease

Article Title: Ubc9 regulates the expression of MHC II in dendritic cells to enhance DSS-induced colitis by mediating RBPJ SUMOylation

doi: 10.1038/s41419-023-06266-1

Figure Lengend Snippet: A A cut-off was set up (LPS-stimulated group versus unstimulated group) as fold changes for quantitative analysis. GO enrichment-based clustering was applied to analyze the biological process of potential SUMOylated proteins. B Predicted SUMOylation sites in RBPJ. C Assay for the SUMOylation of RBPJ in HEK293T cells transfected with plasmids encoding Flag-tagged WT RBPJ or lysine-to-arginine RBPJ mutant, together with plasmid encoding SUMO1. D SUMOylation analysis using HEK293T cells transfected with the indicated vectors. E RBPJ SUMOylation assay using HEK293T cells co-transfected with WT RBPJ or its triple mutant variants and SUMO1. F WT RBPJ or K110R/K195R mutant RBPJ were individually transfected with SUMO1 into HEK293T cells for RBPJ SUMOylation assay. G Sequence alignment surrounding K110 and K195 in RBPJ from the indicated species. Data are representative of three independent experiments in ( C – F ).

Article Snippet: Antibody against DYKDDDDK Tag (2368S) was obtained from the Cell Signaling Technology (Danvers, MA, USA); antibody against Ciita (A16401) was ordered from Abclonal (Wuhan, China); antibodies against SUMO1 (10329-1-AP) and β-Actin (66009-1-Ig) were originated from Proteintech (Wuhan, China); and antibodies against Ubc9 (sc-271057) and RBPJK (ab25949) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Cambridge, MA, USA), respectively.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Sequencing

Journal: Cell Death & Disease

Article Title: Ubc9 regulates the expression of MHC II in dendritic cells to enhance DSS-induced colitis by mediating RBPJ SUMOylation

doi: 10.1038/s41419-023-06266-1

Figure Lengend Snippet: A Western blot analysis of RBPJ expression in BMDCs before and after LPS stimulation. B RT-PCR results for Rbpj expression in BMDCs treated with vehicle or LPS for 6 h. C , D HEK293T cells were transfected with plasmids expressing Flag-tagged RBPJ in the presence of CHX for indicated time, and Western blot analysis was conducted to analyze the half-life of RBPJ. Proteasome inhibitor MG132 was added in ( D ). E Results of an ubiquitination assay. The Flag immunoprecipitates were probed with an ubiquitin antibody for analysis of RBPJ ubiquitination. F RBPJ ubiquitination assay using WT and KO BMDCs. G A model depicting the transcriptional regulation of MHC class II genes. H RT-PCR analysis of relative mRNA levels of MHC II regulatory factors in BMDCs at stimulated state. I Western blot analysis of Ciita expression in BMDCs after LPS stimulation. J The predicted RBPJ binding sites within the Ciita promoter. K ChIP-PCR results for the analysis of RBPJ binding capability to the Ciita promoter. L ChIP-qPCR was performed for RBPJ in the Ciita promoter in WT and KO BMDCs treated with or without LPS. M Relative luciferase activity in HEK293T cells co-transfected with WT or MU RBPJ and SUMO1. N Dual-luciferase reporter assay performed as in ( M ) in the presence of MG132. O , P BMDCs were transduced with vector, WT RBPJ or Ciita adenovirus as indicated. Results for flow cytometry analysis of MHC class II expression ( O ) and antigen processing capability ( P ). All experiments were repeated independently 3 times. Values were expressed as mean ± SEM. Statistical significance was analyzed by one-way ANOVA in ( O and P ) and by unpaired Student’s t test in other figure parts. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibody against DYKDDDDK Tag (2368S) was obtained from the Cell Signaling Technology (Danvers, MA, USA); antibody against Ciita (A16401) was ordered from Abclonal (Wuhan, China); antibodies against SUMO1 (10329-1-AP) and β-Actin (66009-1-Ig) were originated from Proteintech (Wuhan, China); and antibodies against Ubc9 (sc-271057) and RBPJK (ab25949) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Cambridge, MA, USA), respectively.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Ubiquitin Assay, Binding Assay, Luciferase, Activity Assay, Reporter Assay, Transduction, Plasmid Preparation, Flow Cytometry