Journal: Journal of Hematology & Oncology

Article Title: Intracellular delivery of anti-BCR/ABL antibody by PLGA nanoparticles suppresses the oncogenesis of chronic myeloid leukemia cells

doi: 10.1186/s13045-021-01150-x

Figure Lengend Snippet: The role of Trim-Away pathway in Ab@Tf-Cou6-PLGA NPs-mediated BCR/ABL degradation. a Proteasome inhibitor MG132 (100 nM) treatment weakened the BCR/ABL degradation effects of Ab@Tf-Cou6-PLGA NPs in CML cells. b The suppression effect of Ab@Tf-Cou6-PLGA NPs on BCR/ABL was reduced by TRIM21 knockdown. c Immunofluorescence assay was used to analyze the colocalization of BCR/ABL and TRIM21 after Ab@Tf-PLGA NPs treatment. Scale bar, 10 μm. d Co-IP analysis of the interaction of BCR/ABL and TRIM21 with Ab@Tf-Cou6-PLGA NPs treatment. e Co-IP analysis of the interaction of BCR/ABL and TRIM21 without Ab@Tf-Cou6-PLGA NPs treatment

Article Snippet: Co-Immunoprecipitation (Co-IP) experiments were performed with mouse anti-BCR/ABL antibody (Santa, USA), mouse anti-TRIM21 antibody (Santa, USA) and Protein A/G Magnetic beads (MCE, USA).

Techniques: Knockdown, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 1 TRIM21 attenuates the JEV mediated upregulation of the p-IRF3 level and IFN-β level in human microglial cells. (A) PCR amplification of TRIM21 and TRIM21 (ΔRING) primers was carried out and the product run on 1% agarose gel (upper panel). Expression of wild-type TRIM21 as well as the TRIM21 (ΔRING) domain was confirmed by Western blotting (lower panel). (B) CHME3 cells were transfected with 4 μg of TRIM21 plasmid or TRIM21 (ΔRING) for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21, anti-IRF-3 and anti-β- tubulin antibodies by Western blotting. Representative image is shown. (C) Cells transfected with TRIM21 or TRIM21 (ΔRING) were infected with JEV, and total RNA was isolated post 48 h of transfection. Real-time PCR for IFN-β1 was performed, and an average of three independent sets of experiments is plotted and shown. (D) Luciferase assay for IFN-β for cells transfected with TRIM21 or TRIM21 (ΔRING) and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (*p <0.05, **p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Amplification, Agarose Gel Electrophoresis, Expressing, Western Blot, Transfection, Plasmid Preparation, SDS Page, Infection, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Control

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 4 JEV induces TRIM21 protein levels in human microglial cells in a time-dependent manner. (A) CHME3 cells were either un-infected or infected with JEV at MOI 5 for 6, 12, 24 and 36 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β- tubulin antibody (loading control) by Western blotting. A representation of three independent experiments is shown along with densitometry analysis with normalization of TRIM21 against β-tubulin, averaging and plotting. (B) CHME3 cells were infected with JEV for 24 h (MOI 5). Total RNA was isolated from harvested cells. cDNA prepared by reverse transcription of control and infected samples was used as a template for qPCR against primers for TRIM21 gene. Average fold change in the TRIM21 mRNA level from three independent experiments is plotted and shown (*p < 0.05, **p < 0.01, ***p < 0.001 from control, #p from 6 h, $p from 12 h).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Infection, SDS Page, Control, Western Blot, Isolation, Reverse Transcription

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 5 TRIM21 knockdown facilitates JEV-mediated IRF3 activation and upregulation of the IFN-β level. (A) Cells were either transfected with negative control RNA (NC) or transfected with 10nM siRNA against TRIM21 for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β-tubulin antibody by Western blotting. A representative image is shown. (B) Cells were either non-transfected (C), transfected with negative control RNA (NC) or with TRIM21 siRNA for 24 h followed by JEV infection for 24 h. Cell lysates were resolved on SDS-PAGE and probed with anti-p-IRF3 antibody, anti-IRF3 and anti-β-tubulin antibodies (loading control) by Western blotting. A representative of three independent experiments is shown. Densitometry analyses of Western blot experiments were performed with normalizing p-IRF-3 and p-IRF-3 against β-tubulin. (C) Real-time PCR for IFN-β1 for siRNA-transfected and JEV-infected cells along with the respective controls was performed and averaged for three independent sets of experiments. (D) Luciferase assay for IFN-β for cells transfected with siRNA against TRIM21 and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (**p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Knockdown, Activation Assay, Transfection, Negative Control, SDS Page, Western Blot, Infection, Control, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 6 Model showing a plausible role of TRIM21 as a negative regulator of IRF3 activation and IFN-β production following JEV infection in human microglial cells. JEV infection causes activation of the RIG-1 receptor, initiating a downstream signaling mechanism leading to the activation of IRF-3. Phosphorylated IRF-3 dimerizes and translocates into the nucleus, where it leads to the transcription and production of IFN-β. JEV infection also induces the TRIM21 protein, which negatively regulates IRF-3 phosphorylation, leading to reduced IFN-β production. The upregulation of TRIM21 is proposed to be a feedback mechanism to inhibit the innate immune response in JEV infection.

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Activation Assay, Infection, Phospho-proteomics

Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Tubacin decreases KCTD9 protein and increases KCTD9 K123bhb and K129bhb levels in HEK-293T and different CRC cell lines. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. Cell lysates were immunoprecipitated with anti-KCTD9 and analyzed by WB. B Tubacin has a negligible effect on KCTD9 mRNA levels. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. The relative KCTD9 mRNA levels were determined by qPCR. C , D Immunoblot and qPCR analysis of the KCTD9 K123bhb and K129bhb ( C ), and KCTD9 mRNA levels ( D ) in control and TAF1-knockdown HCT116 and LOVO cells. E Co-IP of endogenous TRIM21 with anti KCTD9 antibody in HCT116 cells. F Effects of TAF1 WT or HAT activity-deficient mutant Δ844-850 on KCTD9 association with TRIM21. Myc-TRIM21 and indicated Flag-TAF1 were co-transfected into HEK-293T cells with HA-KCTD9 constructs or empty vector control (EV). G Flag-tagged TRIM21 protein ubiquitinated HA-tagged KCTD9 analyzed by in vitro ubiquitination assays using purified recombinant proteins. Purified recombinant TRIM21 and TRIM21 ΔRING protein were incubated with ATP, E1, E2 proteins, ubiquitin, KCTD9-WT peptide and KCTD9-Kbhb peptide (10 μM or 20 μM) along with purified recombinant HA-tagged KCTD9 protein. H , I Effects of ectopic expression of KCTD9 WT or KCTD9 2KR mutant on KCTD9 protein degradation in HCT116 cells co-transfection of Myc-TRIM21 and Flag-TAF1 in exposure to cycloheximide (CHX, 100 μM) treatment (0, 30, 60, and 90 min). J Effects of KCTD9 WT, 2KQ or 2KR mutant on KCTD9 ubiquitination in HCT116/sgKCTD9 cells co-transfection of Myc-TRIM21 or/and Flag-TAF1. Data are representative of two or three independent experiments with similar results. Error bars, ± SD. * P < 0.05 or ** P < 0.01, by paired two-way Student’s t-test. n.s., negative significant.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Activity Assay, Mutagenesis, Transfection, Construct, Plasmid Preparation, In Vitro, Ubiquitin Proteomics, Purification, Recombinant, Incubation, Expressing, Cotransfection

Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Representative images of TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD expressions in 113 clinical CRC primary tumor tissues. Scale bars, 50 μm. B Correlation of expression levels between TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD in ( A ). C Prognosis comparison of CRC patients with TAF1/KCTD9-K123bhb, TAF1/KCTD9-K129bhb and TAF1/NICD ectopic differential expression using Kaplan-Meier survival analysis. D A working model of TAF1 promoted CRC metastasis through the KCTD9 Kbhb modification status. TAF1 directly binds to and catalyzes KCTD9 Kbhb modification at lys123 and lys129, thereby enhancing KCTD9 association with the E3 ubiquitin ligase TRIM21. This interaction leads to the promotion of KCTD9 degradation via the ubiquitin-proteasome pathway. KCTD9, as a tumor suppressor, its low levels activated Notch signaling pathway, ultimately contributes to the CRCSC properties, the progression of CRC and liver metastasis.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Expressing, Comparison, Quantitative Proteomics, Modification, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: The sequences of siRNAs used in this study.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Sequencing, Negative Control

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Molecular Weight, FLAG-tag

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A , B HeLa and SiHa cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 1 μM) and proteasome inhibitor MG132 (10 μM). Total cell proteins were collected for western blotting at different time points (0 h, 3 h, 6 h, and 12 h). The addition of MG132 slowed the degradation rate of the NCAPH protein, suggesting the involvement of the ubiquitin proteasome pathway. C Total protein in HeLa cells was immunoprecipitated by agarose beads containing IgG or NCAPH antibodies and silver stained after gel electrophoresis. The protein bands with the most significant differences were subjected to mass spectrometry analysis. D , E Coimmunoprecipitation was used to detect the interaction between TRIM21 and NCAPH. NCAPH and TRIM21 antibodies were used as separate baits; F Immunofluorescence assay showing the colocalization of the NCAPH and TRIM21 proteins in HeLa and SiHa cells. NCAPH, 488 nm, green fluorescence; TRIM21, 594 nm, red fluorescence; nucleus, DAPI, blue fluorescence. G Western blotting analysis showed that interference with NCAPH induced no changes in TRIM21 protein levels in cervical cancer cells. H – J Western blotting showed the efficiency of interference with three sets of siRNAs targeting TRIM21 and a significant increase in NCAPH protein levels after siTRIM21 treatment in HeLa and SiHa cells. *** P < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Staining, Nucleic Acid Electrophoresis, Mass Spectrometry, Immunofluorescence, Fluorescence

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A Construction of the TRIM21 truncation plasmid. Different colored boxes represent different structural domains, while discontinuous points represent deleted structural domains. B , C TRIM21 plasmids and corresponding domain mutants (labeled with HA) were cotransfected with NCAPH plasmids (labeled with Flag) into HEK 293T cells, and co-IP experiments were performed using Flag- or HA-labeled antibodies. Mutations in the PRY/SPRY and CC domains significantly influenced the binding between TRIM21 and NCAPH. D – G Cervical cancer cells were treated with cycloheximide (CHX), and total proteins were collected at different time points. Western blotting analysis revealed that, compared with the control group (siNC), knocking down TRIM21 significantly reduced the degradation rate of the NCAPH protein. H Western blotting analysis showed that MG132 treatment (10 μM) significantly reversed the decrease in NCAPH expression induced by TRIM21 overexpression. I HeLa and SiHa cells were transfected with siTRIM21 and HA-UB plasmids, respectively. Coimmunoprecipitation and western blotting assays showing a significant decrease in the ubiquitination level of NCAPH after interference with TRIM21. J , K HeLa and SiHa cells were transfected with siTRIM21 and cocultured with the HA-UB-K11, HA-UB-K48, or HA-UB-K63 plasmid. Coimmunoprecipitation and western blotting showing the effects of interfering with TRIM21: ubiquitination at the K11 position of NCAPH is significantly decreased. L Western blotting analysis showed that the mutation in the RING domain of TRIM21 resulted in the loss of regulation of NCAPH expression, suggesting that TRIM21 regulates NCAPH expression through its E3 ubiquitin ligase activity. M , N HeLa and SiHa cells were transfected with HA-TRIM21 or HA-TRIM21 Δ RING and cocultured with the His-UB plasmid. Coimmunoprecipitation and western blotting showed that TRIM21 overexpression significantly increased the ubiquitination level of NCAPH, while the elimination of the RING domain of TRIM21 significantly decreased it. The HA pcDNA3.1 vector was used as a control. * P < 0.05.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Plasmid Preparation, Labeling, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Control, Expressing, Over Expression, Transfection, Ubiquitin Proteomics, Mutagenesis, Activity Assay

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A – C Construction of a cervical cancer cell line with stable knockout of TRIM21. HeLa and SiHa cells were transfected with lentivirus for 72 h and then observed under a fluorescence microscope. The cells were screened with puromycin, and the knockdown efficiency was further confirmed by western blotting. Scale = 100 µM. D , E Cervical cancer cells were treated with 5 µM rapamycin for 2–4 h and then with 20 µM chloroquine for 12 h and then observed by transmission electron microscopy. The number of autophagosomes was calculated by quantitative analysis. Results showed that silencing TRIM21 significantly decreased the number of autophagosomes in HeLa and SiHa cells. Data are representative images and means ± SD of 3 fields (100 μm 2 per field). F – H Western blotting analysis showed that TRIM21 silencing decreased the expression of LC3B II and beclin-1 while increasing that of P62. I – L Cervical cancer cells were transfected with TRIM21 siRNA and then stained with AO and LysoTracker Red working solution. Hoechst 33342 (blue) was used to stain the nucleus. Statistical analysis revealed that knocking down TRIM21 had no effect on the fluorescence signals. M – P Western blotting analysis showed that 3-MA (2 mM) treatment almost completely reversed the changes in LC3B II caused by TRIM21 overexpression. In contrast, bafilomycin A1 (100 nM) treatment did not reverse the increase in LC3 II expression caused by TRIM21 overexpression. Q – S Western blotting analysis showed that knocking down TRIM21 expression significantly decreased the expression levels of autophagosome formation-related proteins (Beclin-1, ATG5, and ATG7). shTRIM21 and siTRIM21, TRIM21 interference group; shNC and siNC, negative control group. TRIM21, pcDNA3.1-TRIM21 plasmid; pcDNA3.1, mock vector. Scale = 25 μM. * P < 0.05, ** p < 0.01.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Knock-Out, Transfection, Fluorescence, Microscopy, Knockdown, Western Blot, Transmission Assay, Electron Microscopy, Expressing, Staining, Over Expression, Negative Control, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A – D Immunofluorescence staining was used to detect changes in the expression of LC3B and P62 in HeLa and SiHa cells. Interference with TRIM21 decreased the LC3B signal but increased the P62 signal. Knocking down NCAPH significantly reversed the changes caused by siTRIM21. Scale bar = 50 µm. E – H The mRFP-GFP-LC3 indicator system showed that interference with TRIM21 significantly decreased the spot ratio of mRFP to GFP, while silencing NCAPH dramatically reversed the changes in the spot ratio caused by siTRIM21 treatment. The autophagic flow process was observed and analyzed via confocal microscopy. ImageJ software was used to calculate the number of yellow and red puncta. Scale bar, 10 µm. I , J Western blotting analysis showed that interference with NCAPH eliminated the changes caused by knocking down TRIM21 ( I ). In contrast, overexpression of NCAPH eliminated the increase in LC3B II levels caused by overexpression of TRIM21 ( J ). The figure shows the representative results of three experiments. * P < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Immunofluorescence, Staining, Expressing, Confocal Microscopy, Software, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A , B HeLa and SiHa cells were treated with RAPA (5 μM) for 24 h, and the EdU assay was used to detect cell proliferation capacity. C – F HeLa and SiHa cells were treated with RAPA (5 μM) or 3-MA (25 μM) for 24 h, and colony formation experiments were used to evaluate colony formation ability. G , I HeLa and SiHa cells were transfected with siNCAPH alone or in combination with 3-MA (25 μM). A colony formation assay was used to determine whether 3-MA could rescue the siNCAPH-induced reduction in proliferation. H , J HeLa and SiHa cells were transfected with the NCAPH overexpression plasmid alone or in combination with RAPA (5 μM). A colony formation assay was used to assess whether RAPA could rescue the increase in proliferation induced by ectopic NCAPH expression. K , M HeLa and SiHa cells were transfected with siTRIM21 alone or in combination with RAPA (5 μM) before the colony formation assay was performed. L , N HeLa and SiHa cells were transfected with the TRIM21 overexpression plasmid alone or treated in combination with 3-MA (25 μM) before performing the colony formation assay. The figure shows the representative results of three experiments. siTRIM21, TRIM21 interference group; NC, negative control group. TRIM21, pcDNA3.1-TRIM21 plasmid; pcDNA3.1, mock vector. * P < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: EdU Assay, Transfection, Colony Assay, Over Expression, Plasmid Preparation, Expressing, Negative Control

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A , B Silencing TRIM21 significantly increased the number of colonies of cervical cancer cells, while interference with NCAPH dramatically reversed the changes in colony numbers induced by siTRIM21. C , D The EdU assay was used to detect changes in cell proliferation capacity. Ectopic expression of NCAPH rescues the reduction in cell proliferation induced by the increase in TRIM21 expression. E Cells were treated with siTRIM21 alone or in combination with siNCAPH. Interference with TRIM21 expression increased p-AKT and p-mTOR protein levels but had no significant effect on total AKT or mTOR levels. NCAPH suppression reverses the effects of siTRIM21 on the protein levels of p-AKT and p-mTOR. F – H Western blotting analysis showing that treatment with an AKT/mTOR pathway inhibitor (MK2206) reversed the effects of TRIM21 silencing on the expression of p-AKT, p-mTOR and LC3B. I The proposed model in the study. NCAPH, which is ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy in cervical cancer through AKT/mTOR-dependent signaling. The figure shows the representative results of three experiments. * P < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: EdU Assay, Expressing, Western Blot

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 expression was elevated in acute pancreatitis (AP). (A) Cross‐dataset analysis of TRIM family gene expression using three independent Gene Expression Omnibus (GEO) datasets. (B) Immunohistochemical (IHC) staining for TRIM21 in the pancreas from L‐arginine‐treated and saline‐treated mice (scale bar: 100 µm; n = 6). (C, D) Co‐immunofluorescence of TRIM21 (red) with F4/80 (green; macrophage marker) or MPO (green; neutrophil marker). Cell nuclei were stained with DAPI (scale bar: 25 µm; n = 5). (E) Representative Western blotting images (upper panel) and quantification (lower panel) of the expression levels of TRIM21 protein in circulating monocytes from L‐arginine‐treated and saline‐treated mice ( n = 6). (F) The plasma levels of TRIM21 in healthy controls ( n = 29) and those with mild AP ( n = 41), moderately severe AP ( n = 37), and severe AP ( n = 23). (G) Representative Western blotting images (upper panel) and quantification (lower panel) of TRIM21 levels in circulating monocytes from patients with AP ( n = 12 per subtype) and healthy controls ( n = 12). (H) RT‐qPCR analysis of the mRNA levels of TRIM21 ( n = 5) and (I) Western blotting of the protein levels of TRIM21 ( n = 6) in bone marrow‐derived macrophages (BMDMs) treated with acinar cell supernatants (ACS). Data are expressed as mean ± SEM (B, E–I), and statistical analyses were conducted using Welch's t‐test (B, H), Student's t‐test (E, I), Kruskal‐Wallis test with Dunn's post hoc test (F), and ANOVA with Tukey HSD post hoc test (G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Immunohistochemistry, Saline, Immunofluorescence, Marker, Staining, Western Blot, Clinical Proteomics, Quantitative RT-PCR, Derivative Assay

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage Trim21 deficiency attenuated L‐arginine‐induced AP. (A–G) Trim21 M‐KO and Trim21 M‐WT mice were treated with either saline or L‐arginine for 72 h ( Trim21 M‐WT + Saline, Trim21 M‐WT + L‐arginine, Trim21 M‐KO + Saline, Trim21 M‐KO + L‐arginine; n = 10 per group). (A) Serum amylase levels. (B) Representative gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) Hematoxylin and eosin (H&E) staining (upper panel) and histopathological scoring (lower panel) of the pancreatic tissue. (E) Representative images (upper panel) and quantification (lower panel) of IHC staining for interleukin‐1 beta (IL‐1β) / tumor necrosis factor‐alpha (TNF‐α) in the pancreatic tissue. (F, G) IL‐1β and TNF‐α levels in serum samples and bronchoalveolar lavage fluid (BALF). Data are expressed as mean ± SEM (A, C–G), and statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (A, TNF‐α in E), Welch's ANOVA with Games‐Howell post hoc test (C, IL‐1β in E, F, G), and Kruskal‐Wallis test with Dunn's post hoc test (D).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Saline, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Vilazodone‐mediated activation of TRIM21 exacerbated L‐arginine‐induced AP. (A‐G) Trim21 M‐WT mice pretreated with vehicle or vilazodone intraperitoneally received saline or L‐arginine for 72 h (Vehicle + Saline, Vehicle + L‐arginine, Vilazodone + Saline, Vilazodone + L‐arginine; n = 10 per group). (A) Serum amylase levels. (B) Representative gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) H&E staining (upper panel) and histopathological scores (lower panel) of the pancreatic tissue. (E) Representative images (upper panel) and quantification (lower panel) of IHC staining for IL‐1β and TNF‐α in pancreatic tissue. (F, G) IL‐1β and TNF‐α levels in serum samples and BALF. Data are expressed as mean ± SEM (A, C–G), and statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (A, C), Kruskal‐Wallis test with Dunn's post hoc test (D), and Welch's ANOVA with Games‐Howell post hoc test (E–G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Activation Assay, Saline, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage TRIM21 regulates PHB2‐mediated cGAS/STING signaling pathways. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed proteins (DEPs) in pancreatic tissues from L‐arginine‐induced Trim21 M‐WT and Trim21 M‐KO mice ( n = 3). The bubble diagram represents the number of genes under a specific term. The color of the dots represents the p ‐value. (B, C) Representative Western blotting images and quantification of the expression levels of mitophagy‐associated proteins (PINK1, Parkin, p62, and LC3B) in BMDMs from Trim21 M‐WT and Trim21 M‐KO mice, or Trim21 M‐WT mice transfected with an adenovirus carrying Trim21 (Ad‐ Trim21 ) and then stimulated with ACS for 12 h ( n = 5). (D) Co‐immunoprecipitation (co‐IP) with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis of pancreatic tissues from L‐arginine‐induced AP and saline‐induced control, with anti‐TRIM21 antibody or IgG antibody ( n = 3). (E) Intersection analysis of the DEPs associated with mitophagy and cytosolic DNA‐sensing pathways with the exclusive LC‐MS/MS signature proteins. (F, G) Western blotting of PHB2 in BMDMs from Trim21 M‐WT and Trim21 M‐KO mice, or Trim21 M‐WT mice transfected with Ad‐ Trim21 and then stimulated with ACS for 12 h ( n = 5). (H) Representative immunofluorescence images of mitophagic events in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS. Mitochondria (TOMM20, red) colocalizing with the autophagic marker LC3B (visualized using adenoviral mCherry‐GFP‐LC3B fusion protein) were identified as mitophagic events (indicated by the red arrows; scale bar: 10 µm; n = 5). (I) Representative immunofluorescence images of cytosolic mtDNA accumulation in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS. Areas where TFAM (mitochondrial transcription factor A, green) and double‐stranded DNA (dsDNA, red) signals overlapped, but did not co‐localize with mitochondria (TOMM20, yellow) or the nucleus (DAPI, blue), were identified as cytosolic mtDNA (indicated by the red arrows; scale bar: 10 µm; n = 5). (J) RT‐qPCR of cytosolic mtDNA in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS ( n = 5). (K) Representative Western blot images (left panel) and quantification (right panel) of mitophagy‐associated protein levels (PINK1, Parkin, p62, and LC3B) in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice following transfection with PHB2 siRNA (si‐ Phb2 ) and subsequent 12‐h ACS treatment ( n = 5). (L) Representative immunofluorescence images of mitophagic events in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after transfection with Phb2 siRNA (si‐ Phb2 ) and 12 h of treatment with ACS. TOMM20 (red) colocalizing with the autophagic marker LC3B (visualized using adenoviral mCherry‐GFP‐LC3B fusion protein) was identified as a mitophagic event (indicated by the red arrows; scale bar: 10 µm; n = 5). (M) Representative immunofluorescence images (indicated by the red arrows; scale bar: 10 µm; n = 5) and (N) RT‐qPCR ( n = 5) of cytosolic mtDNA in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after transfection with si‐ Phb2 and 12 h of treatment with ACS. Data are expressed as mean ± SEM (B, C, F, G, J, K, N). Statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (indicators other than p62 in B, C, F, G, K, N), Welch's ANOVA with Games‐Howell post hoc test (p62 in B), and Student's t‐test (J).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Protein-Protein interactions, Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Saline, Control, Immunofluorescence, Marker, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 could bind to PHB2 via its coiled‐coil domain and promoted K11‐linked ubiquitination‐dependent proteasomal degradation. (A) Co‐IP with anti‐TRIM21 (left) or anti‐PHB2 (right) in BMDMs (n = 5). (B) Co‐localization of TRIM21 (red) with PHB2 (green) in BMDMs. Cell nuclei were stained with DAPI (blue) (scale bar: 10 µm; n = 5). (C) Domain diagrams of TRIM21 WT and deletion mutants. (D) Co‐IP analysis of the interactions of Flag‐PHB2 with His‐TRIM21 (WT or mutants) in HEK293T cells ( n = 5). (E) Representative Western blotting images (left panel) and quantification (right panel) of PHB2 levels in BMDMs transfected with gradient concentrations of Ad‐ Trim21 ( n = 5). (F) Representative Western blotting images (upper panel) and quantification (lower panel) of PHB2 levels in Ad‐ Trim21 ‐expressing BMDMs pretreated with chloroquine (CQ; 50 µ m ), 3‐methyladenine (3‐MA; 10 m m ), and Z‐Leu‐Leu‐Leu‐al (MG132; 10 µ m ) for 12 h ( n = 5). (G) Representative Western blotting images (upper panel) and quantification (lower panel) of PHB2 levels in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice treated with cycloheximide (CHX; 100 µg/mL) or MG132 (10 µ m ) for 0 h or 12 h ( n = 5). (H) Co‐IP results for PHB2 ubiquitination levels. BMDMs from Trim21 M‐WT or Trim21 M‐KO mice were treated with MG132 (10 µ m ) for 12 h and with ACS for 12 h ( n = 5). (I) Co‐IP results for exogenous PHB2 ubiquitination levels. HEK293T cells were transfected with the control vector, His‐TRIM21(WT), Flag‐PHB2, and MYC‐ubiquitin (WT/K11/K48/K63) ( n = 5). Data are expressed as mean ± SEM (E–G). Statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (E, F) and Kruskal‐Wallis test with Dunn's post hoc test (G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Staining, Western Blot, Transfection, Expressing, Control, Plasmid Preparation

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage Phb2 deficiency reversed Trim21 deficiency‐mediated improvement in AP. (A–H) Trim21 M‐KO and Trim21 M‐WT mice received AAV9‐sh Phb2 or AAV9‐sh NC for 4 weeks, followed by 72 h of infusion of L‐arginine ( Trim21 M‐WT + AAV9‐sh NC + L‐arginine, Trim21 M‐WT + AAV9‐sh Phb2 + L‐arginine, Trim21 M‐KO + AAV9‐sh NC + L‐arginine, Trim21 M‐KO + AAV9‐sh Phb2 + L‐arginine, n = 10 per group). (A) Serum amylase levels. (B) Gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) H&E staining (upper panel) and histopathological scores (lower panel) of the pancreas. Representative images (E) and quantification (F) of IHC staining for IL‐1β and TNF‐α in the pancreatic tissue. (G, H) Serum and BALF levels of IL‐1β and TNF‐α. Data are expressed as mean ± SEM (A, C, D, F–H), and statistical analyses were conducted using Welch's ANOVA with Games‐Howell post hoc test (A, F), ANOVA with Tukey HSD post hoc test (C, G, H), and Kruskal‐Wallis test with Dunn's post hoc test (D).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 suppression mitigated L‐arginine‐induced development of AP. (A–I) Trim21 M‐WT mice pretreated with vehicle or quisinostat intraperitoneally received L‐arginine for 72 h (AP, quisinostat + AP; n = 10 per group). (A) Serum amylase levels. (B) Gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. H&E staining (D) and histopathological scores (E) of the pancreas. Representative images (F) and quantification (G) of IHC staining for IL‐1β and TNF‐α in pancreatic tissue. (H, I) IL‐1β and TNF‐α levels in serum samples and BALF. Data are expressed as mean ± SEM (A, C, E, G–I), and statistical analyses were conducted using Student's t‐test (A, C, IL‐1β in G, TNF‐α in I), Mann‐Whitney U test (E, H, IL‐1β in I), and Welch's t‐test (TNF‐α in G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Staining, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Proposed working model of TRIM21. Macrophage‐specific TRIM21 upregulation exacerbates the development of AP. Mechanistically, macrophage TRIM21 facilitates PHB2 degradation through ubiquitin‐proteasome processing, leading to cytosolic mtDNA accumulation via impaired PHB2‐mediated mitophagy. This mtDNA homeostasis disruption triggers cGAS‐STING axis activation, thereby exacerbating inflammatory cascades during AP progression.

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Ubiquitin Proteomics, Disruption, Activation Assay

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Schematic workflow of IP-mass spectrometry with HEK293T cells stably expressing FLAG-KRAS G12V or FLAG-RALA G23V . Selected protein hits with more than 10 significant sequence matches and enriched in KRAS G12V lysates by ≥3-fold are shown. An asterisk denotes known KRAS interactors based on BioGRID 4.4 . (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 transfected with EV (denoted as −), GFP-tagged WT, or enzymatically inactive (C54Y) TRIM21. 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots. (D) Western blots and t 1/2 of HA-TPL2 proteins in HEK293T cells stably expressing EV or TRIM21-GFP treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments is quantified with blots from one representative experiment shown. (E) Relative SRE-driven luciferase reporter activity in HEK293T cells transfected with EV or the indicated constructs. Data were pooled from three independent experiments, each done in triplicates, and are presented as mean ± SEM (****p < 0.0001). (F) Western blots and densitometric quantification of the indicated markers in Pa01C and HPAC cells stably expressing a scramble or two different shRNAs against TRIM21 . (G) GSEA plots showing KRAS and MEK oncogenic signatures in TRIM21 -silenced Pa01C cells.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Sequencing, Western Blot, Transfection, Luciferase, Activity Assay, Construct

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) IP-western blots of HEK293T cells stably expressing EV or FLAG-TRIM4 and transfected with EV or TRIM21-GFP. (B) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 cells transfected with a fixed amount of TRIM4 (6 μg in 10 cm 2 plates) and different amounts (1.5 μg, 3 μg, and 6 μg) of TRIM21. (C) Western blots and t 1/2 of endogenous TRIM4 protein in HEK293T cells transfected with the indicated cDNAs and treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments were quantified, with blots from one representative experiment shown. (D) IP-western blots of HEK293T cells stably expressing EV or HA-TPL2 and transfected with EV, TRIM21-GFP, and FLAG-KRAS G12V . (E) IP-western blots showing TRIM21 polyubiquitination in HEK293T cells transfected with EV, TRIM21-GFP, and/or HA-KRAS G12V . For (A), (B), (D), and (E), 48 h after transfection, cells were treated with DMSO or bortezomib (0.1 μM) for 6 h and harvested for western blots.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Volcano plot of KRAS G12V BioID experiments, which identified RNF185 and MYCBP2 as the two E3 ligases enriched by >2-fold through proximal biotinylation by BirA-KRAS G12V . Identification of the known KRAS G12V effectors BRAF and RALA confirmed the robustness of the data. EFR3A was confirmed and published previously. (B) IP-western blots confirming the interaction of HA-KRAS G12V with endogenous RNF185 and TRIM21 in HEK293T cells. (C) IP-western blots of HEK293T cells stably expressing EV or FLAG-tagged KRAS G12D treated with DMSO or MRTX1133 at 0.1 and 0.5 μM overnight and harvested for western blots. (D and E) Representative Duolink PLA fluorescence images and quantification of fluorescent puncta showing in situ interaction between (D) endogenous RNF185 and KRAS or (E) TRIM21-GFP and endogenous RNF185 in Pa01C and HPAC cells treated overnight with MRTX1133. Data presented as mean ± SEM (****p < 0.0001 by one-way ANOVA; six fields per condition were analyzed; scale bars, 20 μm). (F) IP-western blot of HEK293T cells stably expressing EV or HA-KRAS G12V and transiently transfected with TRIM21-GFP or/and FLAG-RNF185. (G and H) Western blots and t 1/2 of endogenous TRIM21 protein in (G) HEK293T FLAG-KRAS G12D cells stably expressing EV or GFP-RNF185 or (H) Pa01C cell stably expressing a scramble or shRNF185, following treatment with CHX (10 μg/mL) for the indicated durations. (I) Western blots and densitometric quantification of the indicated markers in scramble and two different RNF186 -silenced Pa01C cells. (J) Western blots and t 1/2 of endogenous TRIM21 and TRIM4 proteins in HPAC cells pre-treated overnight with DMSO or MRTX1133, followed by CHX (10 μg/mL) for the indicated durations. (K) Western blots and densitometric quantification showing the impact of overnight MRTX1133 treatment on the indicated markers in Pa01C and HPAC cells. For (G), (H), and (J), data from three independent experiments were quantified, with blots from one representative experiment shown.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Western Blot, Stable Transfection, Expressing, Fluorescence, In Situ, Transfection

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet: (A) Workflow of IP-mass spectrometry and enrichment plot demonstrating GSK3β as one of the top hits enriched in HEK293T cells expressing HA-TPL2 plus TRIM21-GFP when compared to HA-TPL2 alone. (B) IP-western blots confirming the interaction of HA-TPL2 with endogenous GSK3β when TRIM21 is overexpressed. (C and D) Bar graphs showing relative luciferase reporter activity in (C) 293T TOPFlash reporter cells transfected with EV or HA-TPL2 and (D) Pa01C and HPAC cells stably expressing a Wnt luciferase reporter treated overnight with TPL2i. (E) Bar graphs showing selected up- and downregulated Molecular Signatures Database (MSigDB) oncogenic signatures with normalized enrichment score and false discovery rate q values. Shown are GSEA plots focusing on Wnt and MEK signatures in MAP3K8 -silenced Pa01C cells. (F and G) Western blots and densitometric quantification of the indicated markers in (F) HEK293T cells stably expressing EV or HA-TPL2 and (G) Pa01C and HPAC cells treated overnight with TPL2i. (H) Relative Wnt luciferase reporter activity in Pa01C and HPAC cells treated with the indicated agents overnight. (I and J) Western blots and densitometric quantification of p-GSK3β and p-β-catenin in (I) Pa01C and HPAC cells stably expressing EV or TRIM21-GFP and (J) scramble or TRIM21 -silenced Pa01C cells. (K) GSEA plot showing the upregulated Wnt pathway signature in TRIM21 -silenced Pa01C cells. (L) IP-western blots showing GSK3β polyubiquitination in HEK293T cells stably expressing the indicated cDNAs. (M) Western blots and t 1/2 of endogenous GSK3β in Pa01C cells stably expressing EV or TRIM21-GFP treated with CHX (10 μg/mL) for the indicated durations. Data from three independent experiments were quantified, with blots from one representative experiment shown. (N and O) Relative TOPFlash luciferase reporter activity in HEK293T cells stably expressing the indicated cDNAs (N) treated overnight with DMSO or GSK3β inhibitor and (O) treated overnight with TPL2i. (P) Relative Wnt luciferase reporter activity in Pa01C and HPAC cells treated overnight with the indicated agents. For (C), (D), (H), and (N)–(P), data were pooled from two or three independent experiments, each done in technical triplicates, and are presented as mean ± SEM. The p values were calculated from two-tailed t test or ANOVA for multiple groups. **** p < 0.0001, *** p < 0.0002, ** p < 0.0021, * p < 0.0332.

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Mass Spectrometry, Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Stable Transfection, Two Tailed Test

Journal: Cell reports

Article Title: The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS

doi: 10.1016/j.celrep.2024.114667

Figure Lengend Snippet:

Article Snippet: pLVX puro TRIM21 C54Y-GFP , Addgene , Cat# 116942.

Techniques: Ubiquitin Proteomics, Recombinant, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Ligation, Western Blot, Over Expression, Plasmid Preparation, Amplification, Clone Assay, Expressing, Software, Microscopy

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Mascot search results of mass spectrometric peptides sequencing of tyrosine phosphorylated proteins following FcγRI cross linking of THP-1 cells (n = 3).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Sequencing, Control, Ubiquitin Proteomics, Binding Assay, Variant Assay, Activation Assay

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: ( A ) Representative immunoprecipitation of THP-1 cell lysates using anti-pTyr mAb (4G10) followed by Western blotting using selected antibodies showed abundant Tyr phosphorylation of FcγR s, clatherin, HSP70, Cbl, HGS and TRIM21 in cells co-ligated with anti-FcγRI+IgG 1 control mAb validating LC-MS/MS data. Importantly, LILRB4 co-ligation with FcγRI markedly reduced Tyr phosphorylation of all proteins except for HSP70 (n = 3). ( B ) Summary of densitometry of bands from 3 independent experiments showed significant reduction of FcγRs, clatherin, Cbl, HGS and TRIM21 phosphorylation, but not HSP70, in THP-1 cells co-ligated with anti-FcγRI and anti-LILRB4 mAbs, compared to cells co-ligated with anti-FcγRI and negative control mAb (n = 3, **p < 0.01; ***p < 0.001). ( C ) Representative Western blotting of total cell lysates showed that co-ligation of FcγRI with LILRB4 did not alter the total amounts of any of the above proteins when compared to co-ligation of FcγRI+IgG 1 control, ligation of LILRB4 alone or treatment with IgG 1 control alone; the lower panel is the same membrane stripped and re-probed with anti-β actin Ab, confirming comparable protein loading. ( D ) Summary of densitometry analysis of 3 independent experiments showed no significant differences in total FcγRs, clatherin, HSP70, Cbl, HGS and TRIM21 in THP-1 cells within the 4 different treatment groups (n = 3). Full image of the Western blots is shown in . ( E ) Western blotting of cell lysates from FcγRI cross-linked cells showing increased Tyr-phosphorylated Syk that was markedly reduced upon co-ligation with LILRB4, confirming our earlier finding and validating current LC-MS/MS data (n = 1).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Control, Liquid Chromatography with Mass Spectroscopy, Ligation, Negative Control, Membrane

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Cross-linking of FcγRI by immune-complexes causes Tyr phosphorylation of the ITAMs of its common γ chain and binding of pSyk transduces activating signals. This simultaneously initiates phosphorylation of clatherin that causes lateral diffusion of receptor-ligand complexes to clathrin-coated pits, membrane invagination and generation of clathrin-coated vesicles, and/or initiates phosphorylation of Cbl that may directly ubiquitinate the receptor. Phosphorylated Cbl triggers phosphorylation of HSP70 that facilitates un-coating of the vesicles, a precondition for vesicles to fuse with early endosomes and release ligands. The released receptors are transported to either the late endosome and/or lysosome for proteosomal and/or lysosomal degradation or are recycled to the cell surface. The immune complexes in the endosome are either directly degraded by Cbl, or delivered to the lysosome by phosphorylated HGS-STAM 1/2 complex for final degradation. During transfer, immune complexes that escape the endosome are recognised by phosphorylated TRIM21 for proteasomal degradation. Co-ligation of FcγRI with LILRB4 may recruit phosphatases such as SHP-1 to its ITIMs that subsequently dephosphorylate (deactivate) the key molecules including clathrin (1), FcγRI and Syk (2), Cbl (3), HGS and STAM 1/2 (4) and TRIM21(5). These effects may reduce cellular activation and/or suppress receptor/ligand endocytosis. *New Tyr phosphorylated and dephosphorylated proteins identified in this study.

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Phospho-proteomics, Binding Assay, Diffusion-based Assay, Membrane, Ligation, Activation Assay

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a Western blots and densitometry analysis of Trim21 in splenocytes from female MRL/ lpr mice (9 weeks, n = 4; 17 weeks, n = 4; 23 weeks, n = 5). b Spleen CD19 + B cells from 8-week-old female MRL/ lpr mice were transfected with Trim21 siRNA and stimulated with CD40 ligand and IL-4 for 4 days. Levels of total IgG and IgG2a in the culture supernatant were determined using ELISA. c – k Eight-week-old female MRL/ lpr mice were injected with mock ( n = 4) or Trim21 overexpression ( n = 4) vector for 8 weeks: weights and lengths of spleens ( c ) and percentage of spleen weight relative to body weight ( d ); representative photomicrographs of PAS-stained kidney tissues of MRL/ lpr mice ( e ) with histopathologic analysis ( f ) (in e , glomerular and tubular images (top) and vascular images (bottom) are shown; scale bar, 100 μm); serum levels of anti-dsDNA antibodies (total IgG) in MRL/ lpr mice measured using ELISA ( g ); flow cytometric analysis of double-negative T cells in peripheral blood of 16-week-old MRL/ lpr mice (live T cells were defined as Fixable Viability Dye − CD90.2 + cells) ( h ); flow cytometric analysis of regulatory B cells ( i ) and pDCs or IFNα-producing pDCs ( j ) in splenocytes of 16-week-old MRL/ lpr mice; flow cytometric analysis of GC B cells, plasma B cells and IL-10- or IL-17-producing B cells in peripheral blood of 16-week-old MRL/ lpr mice ( k ). All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( a ), two-tailed paired t -test ( b – d , f and h – k ) or two-way ANOVA ( g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Injection, Over Expression, Plasmid Preparation, Staining, Clinical Proteomics, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a , d Flow cytometric analysis of B cells ( a ) and pDCs ( d ) in splenocytes from WT B6 ( n = 10) and Trim21 −/− ( n = 9) mice. In a , total B220 + cells, GC B cells, plasma B cells and regulatory B cells were analyzed. In d , pDCs and IFNα-producing pDCs were analyzed. b , c Splenic B220 + B cells were stimulated with CD40 ligand, anti-IgM and IL-4. mRNA levels of Blimp1 , Xbp1 , Bcl6 and Pax5 in the cells were determined using qPCR ( b ), and levels of total IgG, IgG1, IgG2a and IgG3 in the culture supernatant were determined using ELISA ( c ). All data are shown as mean ± s.e.m. Statistical analyses were performed using two-tailed paired t -test ( a , b and d ) or two-way ANOVA ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: Eight-week-old female WT B6 and Trim21 −/− mice were treated with vehicle (acetone alone, n = 4) or R848 ( n = 4) for 31 days. a – c Representative photomicrographs of PAS-stained kidney tissues of mice with histopathologic analyses. Glomerular and tubular images ( a ) and vascular images ( b ) are shown (scale bar, 100 μm). d Flow cytometric analysis of GC B cells, plasma B cells and regulatory B cells in splenocytes from mice. e , f Flow cytometric analysis of the STING pathway in splenocytes from mice. The bar graphs represent the relative Mean Fluorescence Intensity (MFI) values analyzed in CD19 + B cells ( e ) or Siglec-H + PDCA1 + Freq (%) of CD11c + DCs ( f ). g , h Confocal images of spleen sections stained for CD19 (green) and Cxcl10 (red) ( g ) or PDCA1 (red) and IFNα (green) ( h ) (scale bar, 20 μm). Cell counts are shown on the right. All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( c and d ) or two-tailed paired t -test ( e – h ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Staining, Clinical Proteomics, Fluorescence, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a Representative images of 7-month-old WT ( Trim21 +/+ ) and Trim21 −/− B6. lpr mice with their spleen (scale bar, 1 cm). b The spleen weights of 7-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 13) B6. lpr mice. c ACR measured from urine of 10-month-old Trim21 +/+ ( n = 4) and Trim21 −/− ( n = 4) B6. lpr mice. d , e Representative photomicrographs of PAS-stained kidney tissues of 7-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 13) B6. lpr mice with histopathologic analyses. Glomerular and tubular image (top) and vascular image (bottom) are shown ( d ; scale bar, 100 μm). f IgG2a measured from serum of Trim21 +/+ and Trim21 −/− B6. lpr mice using ELISA. g Flow cytometric analysis of GC B cells, plasma B cells, regulatory B cells, pDCs and IFNα-producing pDCs in peripheral blood of 5-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 11) B6. lpr mice. h Flow cytometric analysis of GC B cells, plasma B cells, regulatory B cells, pDCs and IFNα-producing pDCs in splenocytes of 7-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 15) B6. lpr mice. i , j Flow cytometric analysis of the STING pathway in splenocytes. The bar graphs represent the relative MFI values analyzed in CD19 + B cells ( i ) or Siglec-H + PDCA1 + Freq (%) of CD11c + DCs ( j ). All data are shown as mean ± s.e.m. Statistical analyses were performed using two-tailed paired t -test ( b , c , e and g – j ) or two-way ANOVA ( f ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ACR albumin/creatinine ratio.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a HEK293 cells were immunoprecipitated with an anti-STING antibody or normal IgG, and immunoblotted using anti-TRIM21 and anti-STING antibodies. b NIH3T3 cells were transfected with mock or mouse Trim21 overexpression vector (3 μg) with MG132 (5 μM) treatment. The cells were immunoprecipitated with anti-Sting antibodies or normal IgG and immunoblotted using anti-ubiquitin, anti-Sting and anti-Trim21 antibodies. c In vitro ubiquitination assay using recombinant TRIM21 and STING proteins. The reaction mixture was incubated at 37 °C for 1 h. Proteins were separated by SDS–PAGE and immunoblotted using anti-ubiquitin, anti-TRIM21 and anti-STING antibodies. d PLA to detect protein interactions between mCherry and Sting in NIH3T3 cells overexpressing pmCherry-C1- Trim21 , pmCherry-C1- Trim21ΔRING-Box and pmCherry-C1- Trim21ΔPRYSPRY . Nuclei were stained with DAPI (scale bar, 20 μm). e NIH3T3 cells were transfected with pmCherry-C1 mock or Trim21 overexpression vectors and treated with MG132 (5 μM). Cells were immunoprecipitated with anti-Sting antibodies and immunoblotted with anti-ubiquitin, anti-Sting and anti-mCherry antibodies. f Western blots and densitometry analysis of cells for Sting and Trim21. NIH3T3 cells were treated with MG132 (5 μM) or NH 4 Cl (10 mM). g Western blots and densitometry analysis of splenocytes from WT B6 and Trim21 −/− mice for Sting and Trim21. h Splenocytes from WT B6 and Trim21 −/− mice were stimulated with H-151 for 2 days. IFNα-producing pDCs in the cells were analyzed using flow cytometry. All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( d , f and h ) or two-tailed paired t -test ( g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Immunoprecipitation, Transfection, Over Expression, Plasmid Preparation, Ubiquitin Proteomics, In Vitro, Recombinant, Incubation, SDS Page, Staining, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a HEK293 cells were transfected with mock or human TRIM21 overexpression vector (3 μg) with MG132 (5 μM) treatment. The cells were immunoprecipitated with anti-STING antibodies or normal IgG, and immunoblotted using anti-ubiquitin, anti-STING and anti-TRIM21 antibodies. b Western blots and densitometry analysis of the cells for STING and TRIM21. HEK293 cells were treated with MG132 (1 μM) or NH 4 Cl (1 μM). c , d Western blots ( c ) and densitometry analysis ( d ) of PBMCs from HCs ( n = 7) and patients with SLE ( n = 7) for TRIM21 and STING. e qPCR of PBMCs from HCs ( n = 26) and patients with SLE ( n = 58) for TRIM21 . f Confocal images of PBMCs from HCs ( n = 8) and patients with SLE ( n = 8) for TRIM21 (green), STING (red) and IFNα (white) with DAPI (blue) (scale bar, 20 μm). g , h Stained cell counts of TRIM21 + cells, STING + cells, and IFNα + cells ( g ) or TRIM21 + STING − cells, TRIM21 − STING + cells, and TRIM21 − IFNα + cells ( h ). i , j Correlation between stained cell counts in patients with SLE. Correlation of TRIM21 + cell counts and STING + cell counts ( i ) or TRIM21 + cell counts and IFNα + cell counts ( j ). k , m Flow cytometric analysis of TRIM21 and STING in PBMCs from HCs ( n = 4) and patients with SLE ( n = 4). The bar graphs represent the MFI values analyzed in CD19 + B cells ( k ) and CD11c + DCs ( m ). l qPCR of PBMCs from HCs ( n = 13) and patients with SLE ( n = 26) for CXCL10 . n qPCR of PBMCs from HCs ( n = 13) and patients with SLE ( n = 58) for IFNA2 . All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( b ), two-tailed paired t -test ( d , e , g , h and k – n ) or Pearson’s correlation analysis ( i and j ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Staining, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a , b Correlations between TRIM21 mRNA ( a ) and TRIM21 protein ( b ) expression levels in PBMCs and disease activity-related clinical parameters, including white blood cell (WBC) counts, complement (C3 and C4) levels, anti-DNA antibody levels and SLE disease activity index (SLEDAI) in patients with SLE ( n = 21). TRIM21 mRNA and TRIM21 protein expression were measured by qPCR and western blots, respectively. c , d Correlations between STING1 mRNA expression levels in PBMCs and IFNA2 mRNA expression levels ( c ) and anti-DNA antibody levels ( d ) in patients with SLE ( n = 31). STING1 and IFNA2 mRNA expression were measured by qPCR. e Correlations between activated STING (MFI values of p-STING relative to total STING) and TRIM21 mRNA expression levels in PBMCs of patients with SLE ( n = 38). The expression of STING and p-STING was measured by flow cytometry. f , g mRNA expression levels of TRIM21 ( f ) and IFNA2 ( g ) in PBMCs from anti-TRIM21 antibody-positive ( n = 6) and antibody-negative ( n = 14) patients with SLE measured by qPCR. All data are shown as mean ± s.e.m. Statistical analyses were performed using Pearson’s correlation analysis ( a – e ) or two-tailed paired t -test ( f and g ). * P < 0.05.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Expressing, Activity Assay, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Science advances

Article Title: CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

doi: 10.1126/sciadv.abo7621

Figure Lengend Snippet: Fig. 2. Identification of TRIM21 as a ligand of CLEC-1. (A) Representative affinity chromatography elution products (SDS–polyacrylamide gel electrophoresis) after co-IP of UV-treated Raji or Hpb-all whole-cell extract (WCE) with protein G beads coupled to human Fc CLEC-1 or Fc alone (control) (three independent experiments). Arrowheads at 50 kDa indicate a band only present under the Fc CLEC-1 condition. MW, molecular weight. (B) LC-MS analysis (of the 50-kDa gel band) depicting the top hits of Fc CLEC-1 over the Fc alone control condition (n = 4, mean). (C) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji WCE (or without cells as control) with protein G beads coupled to human Fc CLEC-1 or irrelevant Fc CLEC7A (three independent experiments). Arrowheads correspond to TRIM21 detected in the co-IP in the retained and unretained fractions. (D) Representative Western blot of human CLEC-1 in elution product after co-IP of UV-treated Raji WCE and His–CLEC-1 with protein G beads coupled to antihuman TRIM21 mAb and revealed with either anti-His or anti–CLEC-1 mAb (three independent experiments). (E) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji transduced with lentiviral TRIM21 (sh+) or control scrambled shRNA with protein G beads coupled to human Fc CLEC-1 (three independent experiments). (F) Flow cytometry analysis of human Fc CLEC-1 binding on UV-treated Raji transduced with lentiviral TRIM21 or control scrambled shRNA (Ctrl) (data expressed as fold change of binding of Fc CLEC-1 compared to binding of Fc alone) (n = 5, paired t test, *P < 0.05). (G) Sensograms generated by Biacore-based affinity measurement representing interaction of chip-immobilized recombinant human TRIM21 with five increasing concentrations of human Fc CLEC-1 (Fc alone as control) or His–CLEC-1 [irrelevant (irr) protein as control]. (H) Representative ELISA [density of optical (DO)] of recombinant human TRIM21 on coated human Fc CLEC-1 or Fc alone (n = 4, means ± SEM, unpaired t test, **P < 0.01 and ***P < 0.001).

Article Snippet: Reverse co-IP was performed as co-IP, except that beads were coated with anti-TRIM21 antibody (R&D Systems) or control isotype (BD Pharm) and coincubated with His CLEC-1 protein (Bio-Techne).

Techniques: Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Co-Immunoprecipitation Assay, Control, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transduction, shRNA, Flow Cytometry, Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

doi: 10.1126/sciadv.abo7621

Figure Lengend Snippet: Fig. 6. Treatment with antihuman CLEC-1 antagonist mAb enhances antitumor immunity in CLEC-1 humanized mice. (A) CLEC1A or Clec1a mRNA expression in different subsets of myeloid cells from human or mouse lung tumors from Open-source data of scRNA-seq. AU, arbitrary units. (B) Two-dimensional Uniform Manifold Approximation and Projection and violin plot representations of CLEC1A expression (red scale projection) among clusters of myeloid cells and DCs from scRNA-seq data- set of pan-cancer (breast, colorectal, lung, and ovarian) TME. (C) Box plots representation of log2 [transcript count per million (TMP)+1] values for TRIM21 expression in dif- ferent cancer types from TCGA analyzed by GEPIA tool. BRCA, breast-invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower-grade glioma; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; READ, rectum adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors. Unpaired t test, *P < 0.05. (D and E) Tumor growth or survival curve of CLEC-1 humanized mice with subcutaneous MC38 adenocarcinoma in combination with CPA (150 mg/kg) (D) or with Hepa 1.6 LIHC (E) and treated with irrelevant isotype control or antihuman CLEC-1 antagonist mAbs #1 or #2 (100 g per mouse) twice a week from days 4 to 21 (n = 14 to 17 of two independent experiments, unpaired t test, *P < 0.05 for tumor growth, and log-rank test, *P < 0.05 and **P < 0.01 for survival). (F) Percentage of PMNs/MDSCs (CD11b+Ly6G+MHC-II−), macrophages (CD11b+F4/80+Ly6C+), DCs (CD11c+MHC-II+), CD69+or Tmem (CD44+) CD8+, or CD4+ (CD3+CD4/8+) T cells evaluated by flow cytometry at day 13 after LIHC in the tumor- burdened livers of CLEC-1 humanized mice treated with isotype control or antihuman CLEC-1 antagonist mAbs #4 (100 g per mouse) (data are expressed in the percentage of CD45+ or in MFI of MHC-II expression) (n = 8 to 13, means ± SEM of three independent experiments, unpaired t test *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: Reverse co-IP was performed as co-IP, except that beads were coated with anti-TRIM21 antibody (R&D Systems) or control isotype (BD Pharm) and coincubated with His CLEC-1 protein (Bio-Techne).

Techniques: Expressing, Control, Flow Cytometry