Journal: Theranostics

Article Title: The N 6 -methyladenosine-mediated lncRNA WEE2-AS1 promotes glioblastoma progression by stabilizing RPN2

doi: 10.7150/thno.74600

Figure Lengend Snippet: WEE2-AS1 stabilizes RPN2 protein by preventing CUL2-mediated ubiquitin-proteasome degradation. A qRT-PCR assays showing the relative expression in GBM cells transfected with sh-NC or sh-WEE2-AS1. Data represent the mean ± SD from at least three independent experiments. B Western blot assays showing RPN2 protein levels in GBM cells transfected with sh-NC or sh-WEE2-AS1. Western blot assays showing RPN2 protein level in GBM cells ( C ) transfected with ov-NC or ov-WEE2-AS1, treated with the protein synthesis inhibitor cycloheximide (CHX, 25 μg/mL) at the indicated time, and ( D ) transfected with sh-NC or sh-WEE2-AS1, treated with the proteasome inhibitor MG132 (5 μmol/L) at the indicated time. E Co-IP assays showing the RPN2 ubiquitination levels in GBM cells transfected with sh-NC or sh-WEE2-AS1. UB, ubiquitination. F Top, the potential ubiquitination sites of RPN2 predicted via BDM-PUM ( http://bdmpub.biocuckoo.org/ ) and the UbiBrowser database ( http://ubibrowser.ncpsb.org/ ). Bottom, Co-IP assays showing the RPN2 ubiquitination level after transfection with Flag-tagged wild-type or mutant RPN2 KR vectors. KR, mutation of lysine (K) to arginine G Co-IP assays showing the RPN2 ubiquitination level in GBM cells co-transfected with sh-NC or sh-WEE2-AS1 and Flag-tagged wild-type or K322 mutant RPN2 KR vectors. H Top, Crystal structure of RPN2 proteins with K322. Bottom, conservation ability of the K322 ub site on the RPN2 protein. I Silver staining assays showing the proteins that interacted with RPN2, which were identified by co-IP/mass spectrometry; arrows indicate CUL2 protein bands. J Immunofluorescence staining experiments showing the colocalization of RPN2 and CUL2 in GBM cells. Scale bar, 25 μm. K Western blot assays showing CUL2 and RPN2 protein levels in GBM cells transfected with si-NC or si-CUL2. L Co-IP assays showing the RPN2 ubiquitination levels in GBM cells transfected with si-NC or si-CUL2. M Co-IP assays showing the intensity of the interaction between RPN2 and CUL2 in GBM cells transfected with ov-NC or ov-WEE2-AS1 and treated with MG132 at the indicated times. The statistical significance is shown as follows: ns>0.05. N Western blot assays showing CUL2 and RPN2 protein levels in GBM cells transfected with sh-NC or sh-WEE2-AS1.

Article Snippet: Primary antibodies to the following were used: GAPDH (Cell Signaling Technology, 5174), β-actin (Cell Signaling Technology, 14074), N-cadherin (Cell Signaling Technology, 13116), E-cadherin (Proteintech, 20874-1-AP), CD44 (Proteintech, 15675-1-AP), AKT (Cell Signaling Technology, 9272), phospho-Akt (Ser473, Cell Signaling Technology, 4060), CDK1/cdc2 (Cell Signaling Technology, 9116), phospho-CDK1/cdc2 (Tyr15) (Cell Signaling Technology, 4539), cyclin B1 (Cell Signaling Technology, 12231), P21 (Cell Signaling Technology, 2947), IGF2BP3 (Abcam, ab177477), ubiquitin (Cell Signaling Technology, 3936), DYKDDDDK Tag (Cell Signaling Technology, 14793), His-tag (Cell Signaling Technology, 12698), CUL2 (Proteintech, 10981-2-AP; Santa, sc-166506), and RPN2 (Abcam, ab244399).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Silver Staining, Mass Spectrometry, Immunofluorescence, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: The basic canyon of cullins binds to CSN2. (A–C) Structure of Cul1 (A) (PDB ID code 1LDK) (15) and homology models of Cul2 (B) and Cul3 (C) shown in surface mode. The circled blue region is the basic canyon. (D) Sequence alignment of human Cul1–5. Residues highlighted in red are part of the basic canyon. (E) Sequence alignment Cul4A/B or the single Cul4 from different species. At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis. (F) Lysines 465 and 675 (blue) are located in the groove the canyon. (G) In vitro binding of wildtype K465E or K675E mutants of Cul4A to recombinant CSN2. (H) Myc immunoprecipitation of Cul4A wild-type, the K661/663E mutant.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: Sequencing, In Vitro, Binding Assay, Recombinant, Immunoprecipitation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: IP6 stimulates CSN2–Cul4A binding in vitro. (A) IP6 and IP7 stimulate in vitro binding between HEK293-purified Cul4A and recombinant holo-signalosome. GST-Cul4A pulldown was blotted for CSN1, -2, and -5 simultaneously. (B) IP6 and IP7 stimulate in vitro binding between purified Cul4A and recombinant CSN2. (C and D) IP6 stimulates binding between recombinant CSN2 and HEK293-purified Cul1 (C) and Cul2 (D). (E) Concentration-dependent analysis of IP6 in stimulating CSN2–Cul4A binding. (F) CSN2 concentration-dependent analysis of CSN2–Cul4A binding with/without IP6.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: Binding Assay, In Vitro, Purification, Recombinant, Concentration Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Basis for metabolite-dependent Cullin-RING ligase deneddylation by the COP9 signalosome

doi: 10.1073/pnas.1911998117

Figure Lengend Snippet: IP6 is a general CRL regulator conserved from yeast to mammals. (A–C) GST pulldown of cellular Cul1 (A), Cul2 (B), Cul3 (C) coexpressed with Rbx1 wild-type or mutants deficient in IP6 coordination. Rbx1 mutations disrupting IP6 coordination also abolish CSN interaction with Cul1–3. (D) GST pulldown of CSN2 wild-type and IP6 binding-defective mutants from cell lysates, CSN2 mutations disrupting IP6 coordination also fail to pull down Cul1–5. (E) Deneddylation of purified Nedd8–Cul3/Rbx1 by recombinant CSN, with/without IP6 at the indicated concentrations. (F) Levels of Cullin neddylation in wild-type and IP5K KO cells. *P < 0.05, **P < 0.01. (G) Coimmunoprecipitation between myc-Cul1 and TAP-Csn2, each tagged at endogenous locus in wild-type and Δipk1 S. pombe strains. Cul–Csn2 interaction is abolished in the Δipk1 mutant. (H) UV sensitivity of a Δcsn2 strain rescued with wild-type Csn2, but not the K70E mutant. **P < 0.01.

Article Snippet: The primary antibodies used were: Cul1, Cul3, Cul4A, CSN5 (Cell Signaling); CSN3, Cul4A (ip) (Abcam); Cul2 Cul5 (Bethyl Laboratories); GAPDH (Roche), IP5K (Abcam), and CSN2 (ProteinTech); Cul4B (ip); Trypsin, TNP, shRNA for CSN2 (id: 1773s21c1), IP5K (#1: 1399s1c1;), and scrambled shRNA were purchased from Sigma.

Techniques: Binding Assay, Purification, Recombinant, Mutagenesis

Journal: Hereditas

Article Title: Identification and validation of USP15 and CUL2 as ubiquitination related biomarker in chronic obstructive pulmonary disease.

doi: 10.1186/s41065-025-00460-1

Figure Lengend Snippet: Fig. 11 qPCR was used to detect the expression of USP15 and CUL2 genes in COPD patients and healthy samples. (A) USP15. (B) CUL2

Article Snippet: The specific antibodies mainly consist of USP15 (Proteintech, China), CUL2 (Proteintech, China) and β-actin (Proteintech, China).

Techniques: Expressing

Journal: Hereditas

Article Title: Identification and validation of USP15 and CUL2 as ubiquitination related biomarker in chronic obstructive pulmonary disease.

doi: 10.1186/s41065-025-00460-1

Figure Lengend Snippet: Fig. 10 GSEA enrichment results of eight hub genes. (A) NEDD8. (B) HIF1A. (C) SKP1. (D) BTRC. (E) CAND1. (F) CUL2. (G) UBE2S. (H) USP15

Article Snippet: The specific antibodies mainly consist of USP15 (Proteintech, China), CUL2 (Proteintech, China) and β-actin (Proteintech, China).

Techniques:

Journal: Hereditas

Article Title: Identification and validation of USP15 and CUL2 as ubiquitination related biomarker in chronic obstructive pulmonary disease.

doi: 10.1186/s41065-025-00460-1

Figure Lengend Snippet: Fig. 12 qPCR and western blot experiments were performed to detect the expression of USP15 and CUL2 in COPD cells and control cells. (A-B) The mRNA expression of USP15 and CUL2. (C) The protein expression of USP15 and CUL2

Article Snippet: The specific antibodies mainly consist of USP15 (Proteintech, China), CUL2 (Proteintech, China) and β-actin (Proteintech, China).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Nutrient restriction protects against valve interstitial cell calcification by upregulating ubiquitin mediated proteolysis

doi: 10.3389/fcvm.2025.1586775

Figure Lengend Snippet: Proteomic analysis shows the upregulated UPS proteins in nutrient-deprived RVICs. (A) Fold change and function of proteins involved in ubiquitin proteasome pathway. (B) IPA network analysis shows altered activation of key UPS pathway proteins including Cul2, Ube2H, SKP1 and Ubiquitin highlighted in blue circle. Functional interconnections between the proteins are shown by arrows and lines. The blue lines represent direct associations with Annexin VI. Dashed lines represent predicted associations. (C–E) Validation and quantification of Cul2 and Ube2h expression by immunoblots. Equal amounts of total protein were loaded in each lane, and all blots were processed in parallel under identical experimental conditions to ensure comparability. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies included those against Runx2 (Abcam, ab236639), Cul2 (Proteintech, 10981-2-AP), Ube2h (Proteintech, 15685-1-AP), OCN (Proteintech, 23418-1-AP), Osterix (Proteintech, 28694-1-AP), β-actin (Proteintech, 66009-1-Ig) and bone sialoprotein (Invitrogen, PA5-79425).

Techniques: Ubiquitin Proteomics, Activation Assay, Functional Assay, Biomarker Discovery, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Mitochondrial MUL1 E3 ubiquitin ligase regulates Hypoxia Inducible Factor (HIF-1α) and metabolic reprogramming by modulating the UBXN7 cofactor protein

doi: 10.1038/s41598-020-58484-8

Figure Lengend Snippet: Expression of the various components of the CRL2 VHL complex in the presence or absence of MUL1 protein. (A) The differential expression of CUL2, VHL, PHD2, Elongin B, Elongin C and RBX1 proteins in HEK293 MUL1(+/+) and HEK293 MUL1(−/−) cells was monitored by SDS-PAGE and Western blot analysis. β-actin antibody was used to verify equal loading in each lane. (B) Graph summarizing the differential modulation of the various components of the CRL2 VHL complex between the HEK293 MUL1(+/+) and HEK293 MUL1(−/−) cells using densitometric analysis of the protein expression data from Figs. and 4A. The scale of the graph is presented as log 5 . (C) HEK293 MUL1(+/+) and HEK293 MUL1(−/−) cells were treated with the neddylation inhibitor MLN4924 (1 μM) for various time point (2, 4 and 8 hours). The expression of UBXN7, HIF-1α, CUL2 as well as neddylated-CUL2 was monitored. (D) Graph represents densitometric analysis of the UBXN7 and HIF-1α protein expression from ( C ) normalized against β-actin. * p < 0.03 vs HEK293 MUL1(+/+) control and # p < 0.001 vs HEK293 MUL1(+/+). Results shown are means ± S.D. of three independent experiments.

Article Snippet: The membranes were incubated with the indicated primary antibodies: MUL1 and UBXN7 rabbit polyclonal antibodies are homegrown and used at 1:5000 dilution, HIF-1α (Bioss Antibodies, 1:2000), p97 (SantaCruz, 1:3000), tubulin (SantaCruz, 1:2000), histone H3 (SantaCruz, 1:500), PHD2 (ProteinTech, 1:2000), GLUT1 (NovusBio, 1:1000), Elongin B (Aviva Systems Biology, 1:500), Elongin C (Aviva Systems Biology, 1:500), RBX1 (Aviva Systems Biology, 1:500), CUL2 (Thermo Fisher, 1:2000), Ub (Life Sensor, 1:1000), SUMO1 (ENZO, 1:1000), HA (Millipore, 1:2000), His-tag (ProteinTech, 1:500) and β-actin (SantaCruz, 1:3000).

Techniques: Expressing, SDS Page, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Nutrient restriction protects against valve interstitial cell calcification by upregulating ubiquitin mediated proteolysis

doi: 10.3389/fcvm.2025.1586775

Figure Lengend Snippet: Calcification is reduced in nutrient-starved RVICS. (A) Quantification of alizarin red staining for RVICs cultured either with complete DMEM media or HBSS supplemented with or without 2.7Ca/2.5Pi for 48 h ( N = 6). (B) Quantification of calcium deposition in RVICs cultured with complete DMEM media or HBSS in the presence of 2.7Ca/2.5Pi for 48 h ( n = 6). (C) Cell viability of RVICs cultured in complete DMEM media in the presence or absence of 2.7Ca/2.5Pi and HBSS in the presence of 2.7Ca/2.5Pi for 48 h using the Alamar blue assay ( n = 6). (D) ATP measurement of RVICs cultured with or without complete DMEM media in the presence or absence of 2.7Ca/2.5Pi and HBSS in the presence of 2.7Ca/2.5Pi for 48 h using the Alamar blue assay ( n = 6). (E) Representative images of immunoblots for RUNX2, BSP and β-actin in RVICs cultured with or without complete DMEM media or HBSS supplemented with 2.7Ca/2.5Pi for 48 h. (F,G) The graphs show the ratios of RUNX2 and BSP to β-actin. (H) Representative images of immunoblots for osterix, OCN and β-actin in RVICs cultured with or without complete DMEM media or HBSS supplemented with 2.7Ca/2.5Pi for 48 h. (I,J) The graphs show the ratios of osterix and OCN to β-actin. Equal amounts of total protein were loaded in each lane, and all blots were processed in parallel under identical experimental conditions to ensure comparability. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies included those against Runx2 (Abcam, ab236639), Cul2 (Proteintech, 10981-2-AP), Ube2h (Proteintech, 15685-1-AP), OCN (Proteintech, 23418-1-AP), Osterix (Proteintech, 28694-1-AP), β-actin (Proteintech, 66009-1-Ig) and bone sialoprotein (Invitrogen, PA5-79425).

Techniques: Staining, Cell Culture, Alamar Blue Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Nutrient restriction protects against valve interstitial cell calcification by upregulating ubiquitin mediated proteolysis

doi: 10.3389/fcvm.2025.1586775

Figure Lengend Snippet: Pharmacological inhibition of Cul2 increases calcification in RVICS. (A) Quantification of calcium deposition in RVICs treated with 50 nM or 100 nM MG132 in complete DMEM media supplemented with 2.7Ca/2.5Pi for 48 h ( n = 3). (B) Representative images of immunoblots for RUNX2, BSP and β-actin in RVICs cultured with or without 2.7Ca/2.5Pi supplemented DMEM media in the presence or absence of 100 nM MG132 for 48 h ( n = 3). (C,D) The graphs show the ratios of RUNX2 and BSP to β-actin. (E) Representative images of immunoblots for osterix, OCN and β-actin in RVICs cultured with or without 2.7Ca/2.5Pi supplemented DMEM media in the presence or absence of 100 nM MG132 for 48 h ( n = 3). (F,G) The graphs show the ratios of osterix and OCN to β-actin. Equal amounts of total protein were loaded in each lane, and all blots were processed in parallel under identical experimental conditions to ensure comparability. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies included those against Runx2 (Abcam, ab236639), Cul2 (Proteintech, 10981-2-AP), Ube2h (Proteintech, 15685-1-AP), OCN (Proteintech, 23418-1-AP), Osterix (Proteintech, 28694-1-AP), β-actin (Proteintech, 66009-1-Ig) and bone sialoprotein (Invitrogen, PA5-79425).

Techniques: Inhibition, Western Blot, Cell Culture

Journal: Frontiers in Cardiovascular Medicine

Article Title: Nutrient restriction protects against valve interstitial cell calcification by upregulating ubiquitin mediated proteolysis

doi: 10.3389/fcvm.2025.1586775

Figure Lengend Snippet: Cul2 knockdown increases calcification in RVICS. (A,B) Representative images of immunoblots for RUNX2, osterix, BSP, OCN and β-actin in RVICs transfected with either scrambled control (SC) or Cul2 siRNA for 72 h ( n = 3) in the presence or abcense of 2.7Ca/2.5Pi. (C–G) The graphs show the ratios of Cul2, RUNX2, osterix, BSP and OCN to β-actin. Equal amounts of total protein were loaded in each lane, and all blots were processed in parallel under identical experimental conditions to ensure comparability. (H) Quantification of calcium deposition in RVICs transfected with either SC or Cul2 siRNA in the presence or absence of 2.7Ca/2.5Pi for 72 h ( n = 3). (I) Quantification of calcium deposition in RVICs transfected with either SC or Cul2 siRNA for 72 h in 2.7Ca/2.5Pi supplemented DMEM media or HBSS. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies included those against Runx2 (Abcam, ab236639), Cul2 (Proteintech, 10981-2-AP), Ube2h (Proteintech, 15685-1-AP), OCN (Proteintech, 23418-1-AP), Osterix (Proteintech, 28694-1-AP), β-actin (Proteintech, 66009-1-Ig) and bone sialoprotein (Invitrogen, PA5-79425).

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Redox Biology

Article Title: Cardioprotective effect of MLN4924 on ameliorating autophagic flux impairment in myocardial ischemia-reperfusion injury by Sirt1

doi: 10.1016/j.redox.2021.102114

Figure Lengend Snippet: Effects of MLN4924 on autophagic flux marker proteins. Protein levels of apoptosis-related proteins (LC3Ⅱ/Ⅰ, P62, Beclin-1, Atg5, LAMP2, Rab7) were also detected (I–O), and (P–S) protein expressions of Nrf2, NQO1 and HO-1 were determined by Western blot analysis. Protein expression levels were normalized against levels of GAPDH, which was used as a loading control. For calculation of relative changes in protein expression or ROS level, values of individual samples were divided by the mean value of samples from the Ad.GFP control group. Data are means ± S.D (n = 5). # P < 0.05vs. Ad.GFP + CON (control cells infected by Ad.GFP). * P < 0.05vs. Ad.GFP + H 2 O 2 (Ad.GFP infected cells treated with H 2 O 2 ). ^ P < 0.05vs. Ad.Sirt1-AS + CON (control cells infected by Ad. Sirt1-AS). & P < 0.05 vs. Ad.GFP + H 2 O 2 +MLN4924.

Article Snippet: The primary antibodies used were polyclonal antibodies against Cul 2 from Abcam (Cambridge, USA); Nrf2, Cul1, 4A, 4B, 5 from Proteintech (1:1000, Proteintech Biotech, Wuhan, China); P62, Atg 5, Beclin-1, LC3I/II, Cul3, HO-1, NQO1 and Sirt1 from Cell Signaling (MN, USA).

Techniques: Marker, Western Blot, Expressing, Control, Infection

Journal: Redox Biology

Article Title: Cardioprotective effect of MLN4924 on ameliorating autophagic flux impairment in myocardial ischemia-reperfusion injury by Sirt1

doi: 10.1016/j.redox.2021.102114

Figure Lengend Snippet: MLN4924 induced autophagic flux and up-regulated Nrf2 in MI/R mice via Sirt1. Cardiac protein levels of LC3Ⅱ/Ⅰ, P62, Beclin-1, Atg5 and Rab7 in Sham and MI/R mice were detected for five independent experiments, respectively (E – K). Effect of MLN4924 on protein levels of Cullin substrate proteins (Nrf2, NQO1, HO-1 and NEDD8 modification of Cullin1, 2, 3, 4A, 4B and 5) (L – P). Data were presented as mean ± S.D. (n = 5). # P < 0.05 vs. Sham group. * P < 0.05 vs. MI/R group.

Article Snippet: The primary antibodies used were polyclonal antibodies against Cul 2 from Abcam (Cambridge, USA); Nrf2, Cul1, 4A, 4B, 5 from Proteintech (1:1000, Proteintech Biotech, Wuhan, China); P62, Atg 5, Beclin-1, LC3I/II, Cul3, HO-1, NQO1 and Sirt1 from Cell Signaling (MN, USA).

Techniques: Modification