uba3  (R&D Systems)

Journal: Advanced Science

Article Title: Neddylation Targets and Stabilizes NLRP3 to Augment Inflammasome‐Mediated Colitis and Mood Disorder

doi: 10.1002/advs.202505906

Figure Lengend Snippet: Neddylation promotes the activation of NLRP3 inflammasome in macrophages. BMDMs from Uba3 ΔMye (a–c) or Nedd8 ΔMye (d–f) mice and their corresponding littermates were primed with 100 ng/mL LPS for 4 h, followed by treatment with 5 m m ATP for 30 min (LPS +ATP) or 200 ng/mL MSU for 6 h (LPS +MSU). (a,d) Supernatants were harvested and subjected to ELISA to detect TNF‐α, IL‐6, and IL‐1β levels ( n = 9/group). (b,e) Total RNA was extracted and subjected to quantitative RT‐PCR of Tnf , Il6 , and Il1b ( n = 3/group). (c,f) Whole cell lysates were harvested and subjected to IB to detect inflammasome activation. Error bars show mean ± SD. P values were determined by two‐way ANOVA. * , nonspecific band; Casp‐1, Caspase‐1. All data in this figure are representative of three independent experiments.

Article Snippet: Uba3 F/F mice were crossed with Cx3cr1‐CreERT2 mice on the C57BL/6 background (Shanghai Model Organisms Center, Inc.) to generate Uba3 F/F; Cx3cr1‐CreERT2 ( Uba3‐ iKO) mice.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Neddylation Targets and Stabilizes NLRP3 to Augment Inflammasome‐Mediated Colitis and Mood Disorder

doi: 10.1002/advs.202505906

Figure Lengend Snippet: Myeloid neddylation blockade renders mice resistant to DSS‐induced colitis. (a–f) Uba3 F/F and Uba3 ΔMye mice ( n = 7/group) were treated with 3% DSS for 7 consecutive days. Body weight was monitored daily. After the disease activity index was calculated, all mice were sacrificed, and the colon tissues were harvested. The experiment schedule (a), body weight changes (b), disease activity index (c), representative images of H & E staining (d, scale bar: 200 µm), pathological scores according to the histology (e), and cytokine levels analyzed by ELISA (f) are shown. (g,h) Flow cytometry analysis of macrophages infiltrating the colon ( n = 4/group) after 4 days of 3% DSS treatment, gating live single CD45 + cells. Representative plots (g) and the number of macrophages (h) are shown. (i,j) IB analysis of pro‐IL‐1β and IL‐1β in the colon after 4 days of 3% DSS treatment (i, n = 3/group) and the corresponding densitometric readings (j). Error bars show mean ± SD. P values were determined by two‐tailed Student's t test (c,e,h) or two‐way ANOVA (b,f,j). * , nonspecific band. All data in this figure are representative of two independent experiments.

Article Snippet: Uba3 F/F mice were crossed with Cx3cr1‐CreERT2 mice on the C57BL/6 background (Shanghai Model Organisms Center, Inc.) to generate Uba3 F/F; Cx3cr1‐CreERT2 ( Uba3‐ iKO) mice.

Techniques: Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: Neddylation Targets and Stabilizes NLRP3 to Augment Inflammasome‐Mediated Colitis and Mood Disorder

doi: 10.1002/advs.202505906

Figure Lengend Snippet: Inducible neddylation blockade in microglia mitigates psychological stress‐induced anxiety‐like behavior. Uba3 F/F and Uba3 ‐iKO mice were subjected to restraint from 0:00 to 8:00 a.m. each day for 8 consecutive days (stress group) or placed in the home cage at the same time without food and water (control group), followed by the open field test (OFT). Then stressed mice were sacrificed. Amygdala coronal sections were prepared, or amygdala tissues were lysed in RIPA buffer. (a) The experiment schedule. (b,c) Representative tracks (b) and statistical data (c, n = 8–17/group) of OFT. (d,e) Immunofluorescence staining of Iba1 and CD68 in amygdala coronal sections of stressed mice ( n = 8/group). Representative images (d, scale bar: 50 µm) and statistical data of Iba1 and CD68 co‐labeling (e) are shown. (f–i) IB analysis of IL‐1β maturation (f) and synaptic loss (h) in the amygdala of stressed mice ( n = 3/group) and the corresponding densitometric readings (g,i). * , nonspecific band; Syn, Synaptophysin. Error bars show mean ± SD. P values were determined by two‐tailed Student's t test (e) or two‐way ANOVA (c,g,i). All data in this figure are representative of two independent experiments.

Article Snippet: Uba3 F/F mice were crossed with Cx3cr1‐CreERT2 mice on the C57BL/6 background (Shanghai Model Organisms Center, Inc.) to generate Uba3 F/F; Cx3cr1‐CreERT2 ( Uba3‐ iKO) mice.

Techniques: Control, Immunofluorescence, Staining, Labeling, Two Tailed Test

Journal: Advanced Science

Article Title: Neddylation Targets and Stabilizes NLRP3 to Augment Inflammasome‐Mediated Colitis and Mood Disorder

doi: 10.1002/advs.202505906

Figure Lengend Snippet: Neddylation maintains the protein level of NLRP3. (a–c) BMDMs from Uba3 F/F and Uba3 ΔMye mice were treated as described in Figure , followed by ROS measurement (a, n = 7/group), IB analysis of upstream components of NLRP3 inflammasome (b), and quantitative RT‐PCR of Nlrp3 (c, n = 3/group). (d,e) BMDMs from Nedd8 F/F and Nedd8 ΔMye mice were treated as stated in Figure , followed by IB of NLRP3 (d) and quantitative RT‐PCR of Nlrp3 (e, n = 3/group). (f–k) The indicated genotypes of mice were treated with 3% DSS for 4 consecutive days (f–h) or with restraint for 8 consecutive days (i–k). Colon tissues (f–h) or amygdala tissues (i–k) were harvested, respectively, followed by IB analysis of NLRP3 (f,i, n = 3/group), densitometric readings of IB blots (g,j), and quantitative RT‐PCR of Nlrp3 (h,k, n = 4/group). (l–n) Clinical non‐colitis control colon tissues ( n = 12) and colon tissues with chronic colitis ( n = 11) were examined for NEDD8 and NLRP3 staining on tissue microarray slides. Representative immunohistochemistry images of paired samples (l, scale bar: 100 µm), comparison of NEDD8 and NLRP3 staining levels between the two groups (m), and Spearman's rank correlation of NEDD8 and NLRP3 staining levels (n) are shown. Error bars show mean ± SD. P values were determined by two‐tailed Student's t test (g,h,j,k,m) or two‐way ANOVA (a,c,e). * , nonspecific band; P‐JNK, phospho‐JNK at Thr183/Tyr185. Data in (a–k) are representative of two independent experiments.

Article Snippet: Uba3 F/F mice were crossed with Cx3cr1‐CreERT2 mice on the C57BL/6 background (Shanghai Model Organisms Center, Inc.) to generate Uba3 F/F; Cx3cr1‐CreERT2 ( Uba3‐ iKO) mice.

Techniques: Quantitative RT-PCR, Control, Staining, Microarray, Immunohistochemistry, Comparison, Two Tailed Test

Journal: Advanced Science

Article Title: Neddylation Targets and Stabilizes NLRP3 to Augment Inflammasome‐Mediated Colitis and Mood Disorder

doi: 10.1002/advs.202505906

Figure Lengend Snippet: Neddylation of NLRP3 antagonizes its ubiquitination and degradation. (a,c) BMDMs from Uba3 F/F and Uba3 ΔMye mice were primed with 100 ng/mL LPS for 4 h in the presence or absence of 20 µ m MG132, followed by treatment with 5 m m ATP for 30 min. The expression of NLRP3 and β‐actin was analyzed by IB (a). NLRP3 ubiquitination and neddylation were examined by IB after IP with an antibody against NLRP3 or control rabbit IgG under partially denaturing conditions (c). (b,d–h) Twenty‐four hours after transfection with the indicated plasmids, HEK‐293T cells were treated with or without MLN4924 (0.2 µ m , 12 h) and MG132 (20 µ m , 6 h) as indicated. Whole cell lysates were harvested and subjected to IB to detect inflammasome activation (b). Myc‐NLRP3 ubiquitination was examined by IB after IP with an antibody against Myc‐tag under partially denaturing conditions (d–h). (i) Twenty‐four hours after HEK‐293T cells were transfected with plasmids encoding His‐NEDD8, Myc‐NLRP3, and GFP‐Trim31, Myc‐NLRP3 neddylation was examined by histidine pulldown. (j) BMDMs from Uba3 F/F and Uba3 ΔMye mice were treated with LPS + ATP. The interaction between endogenous Trim31 and endogenous NLRP3 was analyzed by IB after IP with an antibody against NLRP3 or control rabbit IgG. Densitometric readings normalized with the sum of precipitated NLRP3 and input Trim31 bands. (k) Twenty‐four hours after HEK‐293T cells were transfected with Myc‐NLRP3 WT or K287R and GFP‐Trim31, the interaction between Myc‐NLRP3 and GFP‐Trim31 in was examined by IB after IP with an antibody against GFP. (l,m) BMDMs from Uba3 ΔMye mice were transfected with trim31 siRNA or scramble control siRNA. After 48 h, BMDMs were treated with LPS + ATP. Whole cell lysates were then harvested and subjected to IB. Please note that in (i,k), the amount of Myc‐NLRP3 plasmid in each sample was adjusted to ensure a comparable protein level of NLRP3 between different samples. * , nonspecific band; Ab, antibody; Ub, ubiquitin; WT, wild type; M1, K287R; N8, NEDD8; NC, non‐targeting control; Casp‐1, Caspase‐1. All data in this figure are representative of three independent experiments.

Article Snippet: Uba3 F/F mice were crossed with Cx3cr1‐CreERT2 mice on the C57BL/6 background (Shanghai Model Organisms Center, Inc.) to generate Uba3 F/F; Cx3cr1‐CreERT2 ( Uba3‐ iKO) mice.

Techniques: Ubiquitin Proteomics, Expressing, Control, Transfection, Activation Assay, Plasmid Preparation