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: 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: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 5. TRIM21 induces SUCLG2 degradation through the K63-linkage ubiquitin lysosomal pathway. A) BEAS-2B, A549, HCC827, and H1299 cells were treated with 20 μg mL−1 CHX and collected at the indicated time. The degradation rate of SUCLG2 was tested by western blotting (left-hand panels). Relative SUCLG2 expression was analyzed using ImageJ (right-hand panel). Data represent the average of three independent experiments (mean ± SD). ***p < 0.001. B) CHX (20 μg mL−1) was added to A549 cells for 24 h, and CQ (20 μm) or MG132 (20 μm) was added at the same time. The expression of SUCLG2 was detected using western blotting. C) pcDNA3.1-His-SUCLG2 was overexpressed in A549 cells treated with MG132 (20 μm) or CQ (20 μm) for 24 h. Co-IP and western blotting were used to detect the ubiquitination level of SUCLG2. D) The indicated plasmids were transfected into A549 cells

Article Snippet: SUCLG2 siRNA (SR305801), SIRT5 siRNA (SR323578), and TRIM21 siRNA (SR321894) were ordered from Origene.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 6. The K93-succinylation of SUCLG2 affects the TRIM21-mediated degradation of SUCLG2. A) pcDNA-3.1-His-SUCLG2 was transfected into A549 cells, and co-IP and western blotting were used to detect the succinylation level of SUCLG2. B) The succinylation sites of SUCLG2 predicted by PhosphoSitePlus. C) A549 cells overexpressing the SUCLG2-WT or SUCLG2 lysine residue-mutated plasmids were prepared, and co-IP and western blotting were used to detect the succinylation of protein. D) A549 cells were transfected with the pcDNA-His-SUCLG2 or pcDNA-His-SUCLG2K93R

Article Snippet: SUCLG2 siRNA (SR305801), SIRT5 siRNA (SR323578), and TRIM21 siRNA (SR321894) were ordered from Origene.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Residue

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma.

doi: 10.1002/advs.202303535

Figure Lengend Snippet: Figure 8. Inhibition of succinylation at the SUCLG2 K93 site leads to the mitochondrial dysfunction and reduced tumorigenesis of LUAD cells. A and B) LUAD cell lines A549 (A) and H1299 (B) were transfected with His-SUCLG2 and HA-TRIM21 plasmids. Twenty-four hours later, cells were seeded in 24-well plates. At the indicated times, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The dye was extracted with 10% acetic acid, and the relative proliferation was assessed based on the increase in absorbance at 595 nm (upper panels). Western blotting was used to determine the overexpression efficiency (bottom panels). The data represent the average of three independent experiments (mean ± SD). **p < 0.01, ***p < 0.001. C and D) LUAD cell lines A549 (C) and H1299 (D) were transfected with His-SUCLG2 and Flag-SIRT5 plasmids. Cell growth assays were

Article Snippet: SUCLG2 siRNA (SR305801), SIRT5 siRNA (SR323578), and TRIM21 siRNA (SR321894) were ordered from Origene.

Techniques: Inhibition, Transfection, Staining, Western Blot, Over Expression