Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 is elevated in osteoporotic patients, and its deficiency leads to high bone mass. a Quantitative RT‒PCR analysis of Trim21 mRNA expression in bone specimens from patients with different bone mineral densities (BMDs), which were defined as normal, osteopenia, and osteoporosis. b Correlation analysis between Trim21 mRNA expression and RF-BMD and LS-BMD. RF, right femur; LS, lumbar spine. c Immunoblotting analysis of Trim21 protein expression in the lumbar vertebra of 5-month-old sham-operated or ovariectomized mice. d Schematic diagram showing the analysis of skeletal parameters of mice at different ages. e Alcian blue/Alizarin Red staining of the whole skeleton of 1-week-old Trim21 +/+ , Trim21 +/− , and Trim21 −/− littermates. f X-ray images of Trim21 +/+ and Trim21 −/− mice at 1 month and 6 months (left panel). Quantitative analysis of the tibia length of mice at different ages (right panel). g Representative H&E and S/O staining images of tibial sections from 1-month-old Trim21 +/+ and Trim21 −/− mice (left panel). Quantitative analysis of the growth plate thickness of the indicated mice (right panel). h , i Representative immunofluorescence images ( h ) showing the expression of Sox9 + cells ( i ) in growth plates of tibial sections in 1-month-old Trim21 +/+ and Trim21 −/− mice. j Representative micro-CT images of the proximal tibia bone of 14-week-old mice. Quantitative measurements of bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb.Sp). All bar graphs are presented as the mean ± SD. * P < 0.05; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Micro-CT

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 enhances osteoblast activity and favors bone formation. a , b Representative immunoblotting analysis ( a ) and quantification of Runx2, Osterix, and Trim21 in MC3T3-E1 cells ( b ) treated with osteogenic medium for 0, 4, and 7 days. c Quantitative RT‒PCR analysis of osteogenic biomarker genes ( Osterix, Runx2, and Trim21 ) in OBs with osteogenic induction. d , e Alizarin Red S (upper panel) and ALP (lower panel) staining of primary osteoblasts (OBs) after induction with osteogenic medium for different times ( d ). The percentage of Alizarin Red S- ( n ≥ 3) and ALP- ( n ≥ 3) stained area ( e ). f Quantitative RT‒PCR detection of osteogenic biomarker genes ( Runx2, Osterix, OCN, OPG, and RANKL ) in OBs derived from Trim21 −/ − and Trim21 +/+ mice upon osteogenic induction for 7 days. g , h Representative immunoblotting analysis ( g ) and quantification of Runx2 and Osterix in OBs ( h ) after osteogenic induction for 0, 4, and 7 days. i Representative micro-CT images of calvarial bone defects in 2-month-old Trim21 +/+ and Trim21 −/ − mice after surgical induction for 1 month (left panel). Quantitative measurements of bone volume per tissue volume (BV/TV) and bone defect diameter (right panel). All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Activity Assay, Western Blot, Biomarker Discovery, Staining, Derivative Assay, Micro-CT

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 inhibits osteoclast formation and differentiation. a Representative image of histological sections of the tibia that were stained with TRAP (left panel). Bone marrow (BM) and trabecular bone (TB) are indicated in black. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) were determined (right panel). Scale bar: 100 μm. b Quantification of F-actin ring number in BMM-derived OCs from immunofluorescence staining of Fig. . c Representative immunoblot analysis and quantification of Ctsk expression in BMM-derived OCs from Trim21 +/+ and Trim21 −/− mice. d , e Quantitative RT‒PCR detection of OC differentiation genes ( Ctsk, Nfatc1, Acp5, ATP6vod2, and Mmp9 ) in BMM-derived OCs from Trim21 +/+ and Trim21 − / − mice. PBS indicates PBS containing 30 ng·mL −1 M-CSF, while RANKL indicates induction with 30 ng·mL −1 M-CSF and 100 ng·mL −1 RANKL f , g Quantitative RT‒PCR detection ( f ) of OC differentiation genes ( Ctsk and Nfatc1 ) and TRAP staining ( g ) in BMM-derived OCs from Trim21 f/f mice treated with 30 ng·mL −1 M-CSF plus 100 ng·mL −1 RANKL or PBS containing 30 ng·mL −1 M-CSF for 5 days and infected with lentivirus expressing EGFP or Cre recombinase (defined as LV-Con or LV-Cre, respectively). Quantification of TRAP-positive OCs and the number of nuclei per TRAP + cell (right panel) ( g ). Scale bar: 50 μm. h BMMs derived from 4-week-old Trim21 f/f and Ctsk-cre; Trim21 f/f mice were induced for OC differentiation with either 30 ng·mL −1 M-CSF plus 100 ng·mL −1 RANKL or PBS containing 30 ng·mL −1 M-CSF for 5 days (left panel). Quantification of TRAP-positive OCs and the number of nuclei per TRAP + cell (right panel). Scale bar: 50 μm. i , j Schematic diagram illustrating the coculture model of OCs with BMSCs from Trim21 +/+ and Trim21 − / − mice ( i ). Representative images (left panel) and quantification data of TRAP-positive OCs and nucleus number per TRAP + cell (right panel) ( j ). Scale bar: 50 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Staining, Derivative Assay, Immunofluorescence, Western Blot, Expressing, Infection

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: YAP1/β-catenin signaling is essential for Trim21-mediated osteogenic differentiation. a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in bone marrow mesenchymal stem cells (BMSCs) derived from Trim21 +/+ and Trim21 −/− mice. b Volcano plots of DEPs in BMSCs from Trim21 +/+ and Trim21 −/− mice. c Heatmap analysis of DEPs in BMSCs. Three replicates of each group were included, and the top 29 DEPs are shown. d KEGG enrichment analysis of the DEPs in the BMSCs. e Representative immunoblotting analysis and quantification of BCL9, AXIN1, and YAP1 in BMSCs; proteomics sample: part of the samples subjected to proteomics analysis. f Representative immunoblotting analysis and quantification of BCL9, β-catenin, YAP1, and Runx2 protein expression in BMSCs after osteogenic induction for 7 days. g The endogenous interaction between Trim21, BCL9, β-catenin, and YAP1 was evaluated using a co-IP assay. h Protein‒protein interaction of YAP1 and Trim21 in living cells. The two BiFC plasmids encoding Myc-VN155-YAP1 and HA-VC155-Trim21 along with HA-cerulean were cotransfected into HEK293T cells for 24 h. Representative images showing transfected cells (cerulean) and the interaction between YAP1 and Trim21 (Venus). Nuclei were stained with DAPI. Scale bar: 20 μm. i Immunoblotting analysis of BCL9, β-catenin, YAP1, and HA-Trim21 protein expression in HEK293T cells treated with or without MG132. j Representative images showing the expression of YAP1 + OBs derived from Trim21 +/+ and Trim21 −/ − mice. Scale bar: 20 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 protects mice from lipopolysaccharide (LPS)-induced bone loss. a Quantitative RT‒PCR determination of IL-6 , Osterix , and Runx2 mRNA expression in OBs with or without lipopolysaccharide (LPS) treatment during osteogenic induction. b Schematic diagram showing the H&E staining and micro-CT analysis of Trim21 +/+ and Trim21 − /− mice induced by PBS or LPS. c Representative images of H&E staining of tibia sections of 13-week-old Trim21 +/+ and Trim21 −/− mice induced by PBS or LPS. Bone marrow (BM) and trabecular bone (TB) are labeled with red arrows. d , e Representative micro-CT images ( d ) and BV/TV ( e ) of proximal tibia trabecular bone of 13-week-old Trim21 +/+ and Trim21 −/− mice induced by PBS or LPS. f The bone loss ratio after LPS treatment in global knockout mice (left panel) and conditional knockout mice (right panel) . g Representative micro-CT images and BV/TV of proximal tibia trabecular bone of 13-week-old Trim21 f/f and Ctsk-cre; Trim21 f/f mice induced by PBS or LPS. h Representative micro-CT images of cortical bones and quantification of BV/TV (left panel) and thickness (right panel) of Trim21 f/f and Ctsk-cre; Trim21 f/f mice induced by either PBS or LPS. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Expressing, Staining, Micro-CT, Labeling, Knock-Out

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 orchestrates ovariectomy (OVX)-induced bone metabolism by targeting YAP1 signaling. a Determination of fat cell density in the proximal tibia of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. b Representative micro-CT images and quantitative data (BV/TV) of proximal tibial bone of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. c , d Calcein double labeling of mineral layers of tibial trabecular bone of 5-month-old mice. e Representative images of von Kossa staining of the undecalcified proximal tibia of 5-month-old mice. Scale bar: 50 μm. f IHC staining images of the proximal tibia of 5-month-old mice using an antibody against YAP1. The YAP1-stained positive cells are denoted by the red arrow. Scale bar: 50 μm. g , h Representative images of histological sections of the tibia that were stained with TRAP in Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) was determined. Scale bar: 100 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Micro-CT, Labeling, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: A schematic of Trim21 in the regulation of bone remodeling via YAP1/β-catenin signaling. Normal bone remodeling is maintained by the balance of MSC/osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Trim21, by interacting with the protein complex formed by YAP1/β-catenin/BCL9, dictates the degradation of this protein complex, which in turn inactivates YAP1 and β-catenin signaling, which is essential for osteoblast differentiation. However, Trim21 is critical for maintaining the basic expression of osteoclast biomarkers, including Nfatc1 and Ctsk . Therefore, the coupling of osteoblasts with osteoclasts is attributed to dynamic changes in bone metabolism (left panel). In contrast, the loss of Trim21 causes disassociation with the YAP1/β-catenin/BCL9 complex, which then enters the nucleus for subsequent activation of osteogenic genes, including Runx2 and Osterix . In addition, the loss of Trim21 suppresses the maturation of osteoclasts. Together, these results indicate that Trim21 deficiency alleviates pathological bone loss by activating YAP1/β-catenin signaling

Article Snippet: BMMs were derived from 4- to 8-week-old Trim21 +/+ and Trim21 −/− mice or Trim21 f/f mice, as well as Ctsk-cre; Trim21 f/f mice, and then incubated in α-MEM containing 30 ng·mL −1 M-CSF (MCE, Cat# HY-P70553, USA) for 4 days to generate BMMs.

Techniques: Expressing, Activation Assay

Journal: bioRxiv

Article Title: G 4 C 2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

doi: 10.1101/2020.02.13.947721

Figure Lengend Snippet: Western blots and quantification of Trim21 GFP mediated reduction in POM121 ( A-B ), Nup133 ( C-D ), NDC1 ( E-F ), and GAPDH ( G-H ) in wildtype iPSNs. Student’s T-test was used to calculate statistical significance. * p < 0.05, ** p < 0.01.

Article Snippet: On day 18 of differentiation, 5 x 10 6 iPSNs were nucleofected with 5 μg of antibody and 4 μg Trim21 GFP plasmid DNA (Origene) with Lonza P3 Primary Cell 4D Nucleofector Kit (Lonza) using program DC154.

Techniques: Western Blot

Journal: bioRxiv

Article Title: G 4 C 2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

doi: 10.1101/2020.02.13.947721

Figure Lengend Snippet: ( A-P ) Maximum intensity projections from SIM imaging ( A ) and quantification ( B-P ) of Nup spots and volume in nuclei isolated from wildtype iPSNs following knockdown of Nup133, POM121, NDC1, or GAPDH. Antibody used for Trim21 mediated knockdown as indicated on left, antibodies as indicated on top. N = 3 wildtype iPSC lines, 50 GFP+ nuclei per line/knockdown. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( Q ) Confocal imaging of control iPSNs following Trim21 GFP knockdown of Nup133, POM121, or GAPDH immunostained for Ran. Antibody used for Trim21 knockdown as indicated on left, antibodies as indicated on top. ( R ) Quantification of nuclear to cytoplasmic ratio of Ran. N = 3 wildtype iPSC lines, at least 50 cells per line/knockdown. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. ( S ) Quantification of percent cell death following exposure to glutamate. N = 3 control iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. ( T ) Confocal imaging of cell death in control and C9orf72 iPSNs as measured by propidium iodide (PI) incorporation. Antibody used for Trim Away as indicated on left, glutamate concentration and stain as indicated on top. Scale bar = 5 μm ( A ), 10 μm ( Q ), 100 μm ( T ). * indicates significantly altered Nups.

Article Snippet: On day 18 of differentiation, 5 x 10 6 iPSNs were nucleofected with 5 μg of antibody and 4 μg Trim21 GFP plasmid DNA (Origene) with Lonza P3 Primary Cell 4D Nucleofector Kit (Lonza) using program DC154.

Techniques: Imaging, Isolation, Concentration Assay, Staining

Journal: Cell Death & Disease

Article Title: UBE2S interacting with TRIM21 mediates the K11-linked ubiquitination of LPP to promote the lymphatic metastasis of bladder cancer

doi: 10.1038/s41419-023-05938-2

Figure Lengend Snippet: A The endogenous interaction among UBE2S , TRIM21 and LPP in BCa cells was determined by performing co-IP and western blot assays. Anti- UBE2S (14115-1-AP, Proteintech), anti- TRIM21 (12108-1-AP, Proteintech) and anti- LPP (25045-1-AP, Proteintech) were used for Co-IP (each 2 μg). B Representative immunofluorescence images of UBE2S, TRIM21 and LPP colocalization in the nucleus and cytoplasm of BCa cells. Scale bars: white, 25 μm. DAPI was used to label the cell nucleus. C UBE2S , TRIM21 and LPP protein levels in UBE2S -silenced or UBE2S -overexpressing BCa cells. D TRIM21 and LPP protein levels in TRIM21 -silenced BCa cells.

Article Snippet: Recombinant proteins, including UBE1 (Ag8920, Proteintech), UBE2S (ATAP00745, AtaGenix), TRIM21 (Ag28377, Proteintech), LPP (P1269, FineTest) and ubiquitin (Ag20753 Proteintech), were purchased, while ubiquitin-K11R protein was expressed by a cell-free protein expression kit (PLDK001, PLD technology) according to the manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence

Journal: Cell Death & Disease

Article Title: UBE2S interacting with TRIM21 mediates the K11-linked ubiquitination of LPP to promote the lymphatic metastasis of bladder cancer

doi: 10.1038/s41419-023-05938-2

Figure Lengend Snippet: A LPP protein levels in different groups of BCa cells after treatment with MG132 (10 μM, 12 h) or DMSO. The total ( B , C ) and K11-linked ( D , E ) ubiquitination levels of LPP in UBE2S -knockdown or TRIM21 -knockdown HEK293T cells transfected with corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h). WT wild type. The total ( F ) and K11-linked ( G ) ubiquitination levels of LPP in UBE2S - and TRIM21 -overexpressing HEK293T cells transfected with corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h). H An in vitro ubiquitination assay was performed in the presence of LPP , ubiquitin (Ub), Ub-K11R, UBE1 , UBE2S and TRIM21 . The reaction was analyzed by western blotting with an anti- LPP antibody (25045-1-AP, Proteintech). I Schematic diagram of TRIM21 and its truncated mutant. The total ( J ) and K11-linked ( K ) ubiquitination levels of LPP in full-length TRIM21 and TRIM21 -ΔRING HEK293T cells transfected with the corresponding plasmids (48 h) and treated with MG132 (10 μM, 12 h).

Article Snippet: Recombinant proteins, including UBE1 (Ag8920, Proteintech), UBE2S (ATAP00745, AtaGenix), TRIM21 (Ag28377, Proteintech), LPP (P1269, FineTest) and ubiquitin (Ag20753 Proteintech), were purchased, while ubiquitin-K11R protein was expressed by a cell-free protein expression kit (PLDK001, PLD technology) according to the manufacturer’s instructions.

Techniques: Ubiquitin Proteomics, Knockdown, Transfection, In Vitro, Western Blot, Mutagenesis

Journal: Frontiers in Immunology

Article Title: Isolated anti-Ro52 identifies a severe subset of Sjögren’s syndrome patients

doi: 10.3389/fimmu.2023.1115548

Figure Lengend Snippet: Laboratory and clinical parameters in seropositive Sjögren’s syndrome patients, stratified by anti-Ro52 status.

Article Snippet: Serum anti-Ro52 were purified using Ro52 protein (Arotec Diagnostics, New Zealand)-coupled magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen).

Techniques: Isolation, Comparison, Filtration

Journal: Frontiers in Immunology

Article Title: Isolated anti-Ro52 identifies a severe subset of Sjögren’s syndrome patients

doi: 10.3389/fimmu.2023.1115548

Figure Lengend Snippet: Scatterplots of significant correlations between anti-Ro52 and continuous variables. A linear regression line has been added as well as a goodness-of-fit statistic (R 2 ). ESR , erythrocyte sedimentation rate.

Article Snippet: Serum anti-Ro52 were purified using Ro52 protein (Arotec Diagnostics, New Zealand)-coupled magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen).

Techniques: Sedimentation

Journal: Frontiers in Immunology

Article Title: Isolated anti-Ro52 identifies a severe subset of Sjögren’s syndrome patients

doi: 10.3389/fimmu.2023.1115548

Figure Lengend Snippet: Anti-Ro52 serological subsets in Sjögren’s syndrome patients. Forty-eight patients’ sera were analysed for anti-Ro52 IgG, IgA and IgM via enzyme-linked immunosorbent assay (ELISA). Twenty-five healthy controls (HC) were used to establish the cut-off optical density (OD), defined as mean OD + 2 standard deviations. Dotted lines on the y axis represent the cut-off OD. All statistics are compared to the isolated anti-Ro52 subset (blue), using Kruskal-Wallis and Dunn’s post-hoc test, and Fisher’s exact test for continuous and non-continuous variables respectively. NS , not significant by Kruskal-Wallis tests.

Article Snippet: Serum anti-Ro52 were purified using Ro52 protein (Arotec Diagnostics, New Zealand)-coupled magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: Frontiers in Immunology

Article Title: Isolated anti-Ro52 identifies a severe subset of Sjögren’s syndrome patients

doi: 10.3389/fimmu.2023.1115548

Figure Lengend Snippet: Anti-Ro52 IgG proteomic analyses by mass spectrometry. Five isolated (blue) and ten combined (red) anti-Ro52 Sjögren’s syndrome patients’ anti-Ro52 were analysed according to their heavy (IGHV) (A) and light (IGKV) (B) chain subfamily composition. Frequency of subfamily usage refers to the percentage of patients using the designated subfamily within the isolated or combined anti-Ro52 subsets. Column graphs represent median values. (C) Absolute frequency (count) of amino acid mutations in each IGHV subfamily were quantified for each subset. Each circle represents one subfamily. (D) Repeat frequency analyses were then restricted to the heavy complementarity-determining regions (HCDR) 2 and 3 combined. * p < 0.05. NS , not significant. IMGT , international ImMunoGeneTics information database. Mann-Whitney U test was used for analyses.

Article Snippet: Serum anti-Ro52 were purified using Ro52 protein (Arotec Diagnostics, New Zealand)-coupled magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen).

Techniques: Mass Spectrometry, Isolation, MANN-WHITNEY

Journal: Journal of Clinical Medicine

Article Title: The Toll-like Receptor 7-Mediated Ro52 Antigen-Presenting Pathway in the Salivary Gland Epithelial Cells of Sjögren’s Syndrome

doi: 10.3390/jcm12134423

Figure Lengend Snippet: Increased expression of MHC class I and Ro52 in TLR7-stimulated SGECs. ( A ) Representative images in immunostaining showing the expressions of MHC class I, Ro52 (red), and Ro60 (green) in SGECs from SS patients ( n = 2) stimulated with 1 mM loxoribine for 6 h and 1000 U/mL of IFN-β for 12 h. mIgG1 (green) and NRS (red) were used as isotype controls. White arrowheads: the expression of cytoplasm. Bar: 10 μM. mIgG: mouse IgG, NRS: normal rabbit serum. ( B ) MHC class I and Ro52 and Ro60 signals in SGECs immunoprecipitated by control rabbit IgG or rabbit anti MHC class I antibody from SS patients ( n = 4) stimulated with 1 mM loxoribine for 6 h and/or 1000 U/mL of IFN-β for 12 h analyzed by a Simple Western system. One representative blot is shown. Schemes follow the same formatting.

Article Snippet: The primary antibodies used were rabbit anti-MHC class I polyclonal (Proteintech), rabbit anti-Ro52 polyclonal (Cloud-Clone), and mouse anti-Ro60 monoclonal (Santa Cruz).

Techniques: Expressing, Immunostaining, Immunoprecipitation, Western Blot