Journal: Nature Communications

Article Title: In-situ profiling of glycosylation on single cells with surface plasmon resonance imaging

doi: 10.1038/s41467-025-56390-z

Figure Lengend Snippet: The differential plasmonic images of three lectins ( a 5.56 μM WGA; b 4.54 μM SBA; c 4.90 μM ConA) binding and the fluorescent validation images with fluorescently labeled lectins ( a 2.5 μM WGA-Alexa Fluor 488 conjugate, b ConA-Alexa Fluor 488 conjugate, and c SBA-fluorescein conjugate) binding to different cell lines (from left to right, Hacat, DOK, Cal-27 and HSC-3). And the fluorescent images also show the nuclei of cells stained with 30 μM DAPI. The plasmonic images and fluorescent images are representatives from three experiments, showing similar results. Scale bar: 100 μm for plasmonic images and 20 μm for fluorescent images.

Article Snippet: The detailed information of each cell line is: HeLa (ATCC, CCL2), Hacat (AddexBio, T0020001), Cal-27 (ATCC, CRL-2095), DOK (CancerTools, 153251) and HSC-3 (Sigma-Aldrich, SCC193).

Techniques: Binding Assay, Biomarker Discovery, Labeling, Staining

Journal: Nature Communications

Article Title: In-situ profiling of glycosylation on single cells with surface plasmon resonance imaging

doi: 10.1038/s41467-025-56390-z

Figure Lengend Snippet: a The corresponding binding curves obtained from the plasmonic response (black lines) and the fitted results (colored curves) of three lectins binding to four representative cell lines (from the top to the bottom, WGA, SBA, and ConA, respectively), with gray background indicating cell-to-cell variation. b The density mapping of six different glycans on individual four types of cancer cells. Scale bar: 100 μm. c Significant difference analysis in the density of six glycans among four types of cancer cells, Hacat cells (n = 30), DOK cells (n = 30), Cal-27 cells (n = 30), and HSC-3 cells (n = 30). Data are shown as mean ± SD and were analyzed by unpaired one-way ANOVA with Graphpad Prism 9.5; * P < 0.05; ** P < 0.01; *** P < 0.001, ns not significant. In GlcNAc, all P < 0.001; in Neu5Ac, P DOK < 0.001, P Cal-27 = 0.002 and P HSC-3 < 0.001 ; in GalNAc, P DOK < 0.001, P Cal - 27 < 0.001 and P HSC-3 = 0.92 ; in Gal, P DOK < 0.001, P Cal-27 < 0.001 and P HSC-3 = 0.02; in Man , P DOK < 0.001, P Cal-27 = 0.24 and P HSC-3 < 0.001; in Glc, P DOK = 0.68, P Cal-27 = 0.004 and P HSC - 3 = 0.68. 2D ( d ) and 3D ( e ) PCA score plots of four types of cell lines using the expression densities of six glycans as feature sets. The ellipses and ellipsoids indicate the 95% confidence interval for each data set.

Article Snippet: The detailed information of each cell line is: HeLa (ATCC, CCL2), Hacat (AddexBio, T0020001), Cal-27 (ATCC, CRL-2095), DOK (CancerTools, 153251) and HSC-3 (Sigma-Aldrich, SCC193).

Techniques: Binding Assay, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

doi: 10.1177/0271678X17725211

Figure Lengend Snippet: The adverse effects of silencing endogenous CD200R1 or Dok1 by CD200R1 siRNA or Dok1 siRNA at 24 h after GMH. (a) Representative western blot bands. (b) Quantitative analysis of CD200 showed that protein level of CD200 increased following the treatment with CD200Fc. (c) Quantitative analysis of CD200R1 showed CD200R1 increased in the treatment group and decreased in GMH group. CD200R1 siRNA intervention significantly decreased CD200R1 level. However, CD200R1 level does not change with Dok1 siRNA intervention. (d) Quantitative analysis of Dok1 showed that Dok1 expression significantly decreased by both Dok1 siRNA and CD200R1 siRNA intervention. Data are expressed as mean ± SD, *P < 0.05 vs Sham, #P < 0.05 vs vehicle, &P < 0.05 vs Treatment, n = 6/group, one-way ANOVA followed by the Tukey test.

Article Snippet: Primary antibodies used were CD200 (Santa Cruz Biotechnology), CD200R1 (Santa Cruz Biotechnology), Dok1 (Santa Cruz Biotechnology), IL-1beta (Abcam), and TNF-alpha (Abcam).

Techniques: Western Blot, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

doi: 10.1177/0271678X17725211

Figure Lengend Snippet: Effects of CD200R1 siRNA and Dok1 siRNA on IL-1beta and TNF-alpha expression in the presence of CD200Fc (1.5 mg/kg) at 24 h after GMH. (a) Representative western blots bands. (b) Quantitative analysis of IL-1beta. GMH enhanced while CD200Fc reduced the protein level of IL-1beta. (c) Quantitative analysis of TNF-alpha. GMH enhanced protein level of TNF-alpha while CD200Fc attenuated this induction. Data are expressed as mean ± SD, *P < 0.05 vs Sham, #P < 0.05 vs GMH + Vehicle, &P < 0.05 vs Treatment, n = 6/group, one-way ANOVA followed by the Tukey test.

Article Snippet: Primary antibodies used were CD200 (Santa Cruz Biotechnology), CD200R1 (Santa Cruz Biotechnology), Dok1 (Santa Cruz Biotechnology), IL-1beta (Abcam), and TNF-alpha (Abcam).

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

doi: 10.1101/2025.02.11.636929

Figure Lengend Snippet: A. Transcript-based UMAP visualisation of single cells for T and NK cell subsets, labelled in the corresponding course-level clustering. Data extracted from . B-C. Transcript-based UMAP visualisation of T and NK cell types as defined in (A). Clusters are coloured based on expression density from high to low of SH2D2A (B), DOK2, PTPN6, PTPN11 and VAV1 (C). D. Protein abundance kinetics per cell (LFQ) of SH2D2A, DOK2, PTPN6, PTPN11 and VAV1 from naïve (left) and memory (right) human T cells following anti-TCR stimulation at indicated time points. Data extracted from . DNT: double negative T cell; DPT: double positive T cell; gdT: gamma/delta T cell; MAIT: Mucosal-associated invariant T cell; memCD4 T: memory CD4+ T cell; memCD8 T: memory CD8+ T cell; nCD4 T: naïve CD4+ T cell; nCD8 T: naïve CD8+ T cell; NK_CD16hi: natural killer CD16hi; NK_ CD56hiCD16lo: natural killer CD56hiCD16lo; NK_CD56loCD16lo: natural killer CD56loCD16lo; TCRVb13.1: T cell receptor v/beta 13.1 T cell; TissResMemT: tissue resident memory T cell; Treg: regulatory T cell.

Article Snippet: The following primary antibodies were used: anti-DOK2 (clone E-10, Santa Cruz), anti-pTyr564 PTPN6 (clone D11G5, Cell Signaling Technology), anti-PTPN6 (clone C-19, Santa Cruz), anti-pTyr580 PTPN11 (#3703, Cell Signaling Technology), anti-PTPN11 (clone B-1, Santa Cruz), anti-VAV1 (clone C-14, Santa Cruz Biotechnology), anti-TSAd (clone OTI3C7, Origene), anti-PI3 Kinase p85α subunit (clone AB6, Upstate), anti-GFP (clone B-2, Santa Cruz), anti-pTyr (clone 4G10, Upstate Biotechnology), and anti-GADPH (clone 6C5, Chemicon).

Techniques: Expressing

Journal: bioRxiv

Article Title: Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

doi: 10.1101/2025.02.11.636929

Figure Lengend Snippet: A. JTAg TSAd KO cells were transfected with GFP-tagged TSAd plasmids. After 36 hrs, cells were left unstimulated or activated for 5 min with pervanadate and lysed with digitonin lysis buffer. Lysates were subjected to GFP pulldown and analysed by immunoblotting. In the lysate blot probed with anti-GFP, bands labelled with a red * above, denotes endogenous DOK2 from the previous probing. One representative immunoblot is shown ( n = 2-3). FL=full-length construct. IB=immunoblot. B. Bar graph shows quantification of immunoblotting from A, where signals are normalised to the construct’s GFP signal. The average of the WT TSAd was set to 1 and statistical significance was determined with a one-sample t-test (versus value of 1) ( n = 2-3) C. JTAg cells were transfected with plasmids encoding GFP tagged DOK2, PTPN6 or PTPN11. 5 hrs after transfection, cells were activated with PMA/IO for 16-18 hrs. Cells were rested overnight, then left unstimulated or activated for 5 min with pervanadate and lysed with digitonin lysis buffer. Lysates were subjected to GFP pulldown and analysed by immunoblotting. D-E. Same as in C, but here JTAg cells were transfected with plasmids encoding GFP tagged DOK2 or PTPN11 mutants. A representative immunoblot is shown (D) and the bar graph shows the quantification where signals are normalised to the construct’s GFP signal. Average of the WT construct was set to 1 and statistical significance was determined with a one-sample t-test (versus value of 1) then to WT TSAd sample (E). ( n = 3-4). In the GFP-IP blot probed with anti-GFP, the second band labelled with a red * in the pervanadate stimulated PTPN11 sample, is the PI3K p85α-subunit band from the previous probing. All bar graphs show mean with SD.

Article Snippet: The following primary antibodies were used: anti-DOK2 (clone E-10, Santa Cruz), anti-pTyr564 PTPN6 (clone D11G5, Cell Signaling Technology), anti-PTPN6 (clone C-19, Santa Cruz), anti-pTyr580 PTPN11 (#3703, Cell Signaling Technology), anti-PTPN11 (clone B-1, Santa Cruz), anti-VAV1 (clone C-14, Santa Cruz Biotechnology), anti-TSAd (clone OTI3C7, Origene), anti-PI3 Kinase p85α subunit (clone AB6, Upstate), anti-GFP (clone B-2, Santa Cruz), anti-pTyr (clone 4G10, Upstate Biotechnology), and anti-GADPH (clone 6C5, Chemicon).

Techniques: Transfection, Lysis, Western Blot, Construct

Journal: bioRxiv

Article Title: Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

doi: 10.1101/2025.02.11.636929

Figure Lengend Snippet: A. Schematic of the BiFC assay. B. JTAg TSAd KO cells were transfected with plasmids encoding nYFP and cYFP tagged constructs. After 48 hrs, cells were activated with pervanadate for 5 min, washed, and incubated for 4 hrs. Cells were then prepared for imaging and stained for nucleus and actin. Representative images from the BiFC between TSAd and DOK2, and TSAd and PTPN11 are shown. Co-transfected constructs are noted in the table to the left of the image. Scale bar = 10μM. Merged image of actin (red), YFP (green) and nucleus (blue). Lysates from cells transfected with DOK2 and PTPN11 were subjected to immunoblotting in Fig. S2.

Article Snippet: The following primary antibodies were used: anti-DOK2 (clone E-10, Santa Cruz), anti-pTyr564 PTPN6 (clone D11G5, Cell Signaling Technology), anti-PTPN6 (clone C-19, Santa Cruz), anti-pTyr580 PTPN11 (#3703, Cell Signaling Technology), anti-PTPN11 (clone B-1, Santa Cruz), anti-VAV1 (clone C-14, Santa Cruz Biotechnology), anti-TSAd (clone OTI3C7, Origene), anti-PI3 Kinase p85α subunit (clone AB6, Upstate), anti-GFP (clone B-2, Santa Cruz), anti-pTyr (clone 4G10, Upstate Biotechnology), and anti-GADPH (clone 6C5, Chemicon).

Techniques: Bimolecular Fluorescence Complementation Assay, Transfection, Construct, Incubation, Imaging, Staining, Western Blot

Journal: bioRxiv

Article Title: Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

doi: 10.1101/2025.02.11.636929

Figure Lengend Snippet: TSAd alone, DOK2 alone or both (double KO) were knocked-out in Jurkat E6.1 cells by CRISPR/Cas9 gene editing and phosphorTyr (pTyr) kinetics were monitored. A. Gene edited or WT Jurkat E6.1 cells were serum-starved overnight and left unstimulated (0 min) or stimulated with anti-CD3ε antibodies for 2, 5, 15 or 30 min as indicated. Lysates were resolved on an immunoblot and pTyr (A) was probed for. In 5A, GAPDH was re-probed after pTyr probing. One representative blot from two biological replicates is shown. B. Quantification of pTyr kinetic experiments from A ( n = 2, from two independent experiments). Bar graph shows median with range.

Article Snippet: The following primary antibodies were used: anti-DOK2 (clone E-10, Santa Cruz), anti-pTyr564 PTPN6 (clone D11G5, Cell Signaling Technology), anti-PTPN6 (clone C-19, Santa Cruz), anti-pTyr580 PTPN11 (#3703, Cell Signaling Technology), anti-PTPN11 (clone B-1, Santa Cruz), anti-VAV1 (clone C-14, Santa Cruz Biotechnology), anti-TSAd (clone OTI3C7, Origene), anti-PI3 Kinase p85α subunit (clone AB6, Upstate), anti-GFP (clone B-2, Santa Cruz), anti-pTyr (clone 4G10, Upstate Biotechnology), and anti-GADPH (clone 6C5, Chemicon).

Techniques: CRISPR, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: DOK3 serves as a PRDX1-interacting protein in B cells. (A) Immunoblots were performed in NALM-6 cells upon stable expression of PRDX1, with GAPDH serving as the loading control. (B) A volcano plot depicts differentially expressed proteins identified in co-immunoprecipitation assays of flag-PRDX1 in NALM-6 cells treated with anti-human CD40 antibody, human IL-21, and IL-4 for 48 h. (C) Co-immunoprecipitation of flag-PRDX1 in NALM-6 cells. (D) Representative images of Duolink in situ PLA using rabbit anti-PRDX1, mouse anti-DOK3 antibody, and PLA probes in WT and Prdx1 -OE mouse B cells from the spleen. Scale bar, 50 μm. (E) Quantification of relative intensity of fluorescence in mouse WT and Prdx1 -OE B cells using Duolink in situ PLA ( n = 3). (F) Pull-down assay was performed using HIS-PRDX1 and GST-DOK3 in the purified form. (G) The interaction between His-PRDX1 and AVI-DOK3 was evaluated by BLI. K D represents the dissociation constant ( n = 3). (H) The Line graph shows deuterium uptake plots for peptide 218–237 aa of DOK3 in the presence and absence of PRDX1. (I) The 3D structure of DOK3(1–330 aa) was predicted using Alphafold to illustrate the perturbation of hydrogen–deuterium exchange upon interaction with recombinant PRDX1 protein. Peptide differences with % D between –5% and 5% were considered not significant and are shown in grey, while green–blue represents decreased hydrogen–deuterium exchange rate. Data are plotted as percentage deuterium uptake versus time on a logarithmic scale. (J) The line graph shows the deuterium uptake profile of peptide 43–49 aa of PRDX1 in the presence and absence of DOK3. (K) Differential HDX consolidation view was mapped to the PRDX1 crystal structure (PDB ID: 7WET) to demonstrate hydrogen–deuterium exchange perturbations upon interaction of the recombinant PRDX1 protein and the recombinant DOK3(1–330 aa) protein. (L) Pull-down assay was performed using His-PRDX1mutants (L46A, D47A, F48A, T49A) and GST-DOK3 in the purified form. (M) Relative protein levels of His-PRDX1(Wild type and mutants) in the (L) pull-down assay ( n = 3). Data are shown as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Control, Immunoprecipitation, In Situ, Fluorescence, Pull Down Assay, Purification, Recombinant

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: PRDX1 suppresses plasma cell differentiation by reducing the degradation of DOK3 in the autophagy–lysosome pathway. (A–D) WT B cells were treated with vehicle or siDOK3 and induced with anti-mouse CD40 antibody, murine IL-21, and IL-4. (A) The proportion of plasma cells was analyzed by flow cytometry. (B) A quantitative analysis of plasma cell counts in (A) ( n = 3). (C) Immunoblot analysis was performed to assess the efficiency of DOK3 knockdown and JNK phosphorylation. (D) Relative protein levels of DOK3 and P-JNK in the (C) ( n = 3). (E–H) Representative Western blot showing DOK3, phosphor-JNK, total JNK, and PRDX1 expression in WT, Prdx1- OE (E), and Prdx1 −/− B cells (G) treated with anti-mouse CD40 antibody, murine IL-21, and IL-4 for 5 days, with GAPDH as a loading control. (F, H) Relative protein levels of DOK3, phosphor-JNK in WT, Prdx1 -OE and Prdx1 −/− B cells ( n = 3). (I) Representative images of Duolink in situ PLA using rabbit anti-PRDX1, mouse anti-DOK3 antibody, and PLA probes in B cells from the spleen of the WT and PRDX1 -OE mice, followed by anti-mouse CD40 antibody, murine IL-21, and IL-4. Scale bar, 50 μm. (J) Relative intensity of fluorescence of Duolink in situ PLA in B cells from the spleen of the WT and Prdx1 -OE B cells ( n = 3). (K) WT B cells were stimulated with anti-mouse CD40 antibody, murine IL-2, and IL-4 for the indicated times, and immunoblot analysis was performed to assess DOK3 and JNK phosphorylation. (L) Relative protein levels of DOK3 and P-JNK in WT B cells stimulated with anti-mouse CD40 antibody, murine IL-21, and IL-4 for the indicated times ( n = 3). (M) WT B cells were treated with the lysosome inhibitor CQ following stimulation with anti-mouse CD40 antibody, murine IL-21, and IL-4 for 2 h, and immunoblot analysis was performed to assess DOK3. (N) Relative protein levels of DOK3 in WT B cells treated with the lysosome inhibitor CQ ( n = 3). Data are shown as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Clinical Proteomics, Cell Differentiation, Flow Cytometry, Western Blot, Knockdown, Phospho-proteomics, Expressing, Control, In Situ, Fluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: Salvianolic acid B functions as a molecular glue-like compound enhancing the interaction between PRDX1 and DOK3. (A) The chemical structure of salvianolic acid B is depicted. (B) Pull-down assay of GST-DOK3(1–330 aa) and His-PRDX1 protein incubated with 100, 10, and 1 μmol/L salvianolic acid B, respectively. (C) The binding affinity of AVI-PRDX1 with SAB was measured using a BLI binding kinetic assay. K D stands for the dissociation constant ( n = 3). (D) The binding affinity of AVI-DOK3(1–330 aa) with SAB was measured using a BLI binding kinetic assay. K D stands for the dissociation constant ( n = 3). (E) Electrostatic potential of the SAB–PRDX1 complex crystal structure and the binding site of SAB. The interior of SAB's binding site is electronegative (colored in blue), while its exterior is electropositive (colored in red). SAB was shown in the wheat stick. Unbiased F o – F c density map contoured at 2.5 Å of a crystal structure for PRDX1 in complex with SAB. (F) The hydrogen bond network formed between SAB and residues of PRDX1 is shown. The hydrogen bond network consists of ten hydrogen bonds, displayed as magenta dashed lines, while water molecules are represented as red spheres. (G) HDX analysis of PRDX1 comparing deuterium exchange rates before and after incubation with DOK3(1–330 aa) and after incubation of PRDX1 and DOK3(1–330 aa) with and without the addition of SAB. (H) HDX analysis of DOK3(1–330 aa) was performed to compare the rate of deuterium exchange before and after incubation with PRDX1, as well as the DOK3(1–330 aa) and PRDX1 complexes with SAB versus without SAB. (I) The affinity of the ternary complex of PRDX1, DOK3(1–330 aa), and SAB was measured by BLI binding kinetic assay. K D stands for the dissociation constant ( n = 3). (J) Co-immunoprecipitation of flag-PRDX1 in NALM-6 cells treated with 60 μmol/L SAB is shown. (K) The relative Protein levels of DOK3 in (J) ( n = 3). (L) Pull-down assay was performed using GST-DOK3(1–330 aa) and His-PRDX1 mutant protein (D47A, F48A, T49A) incubated with 50 μmol/L salvianolic acid B. (M) Relative protein levels of His-PRDX1 (Wild type and mutants) in the (L) pull-down assay ( n = 3). (N) The co-immunoprecipitation of flag-PRDX1 and its mutants wih myc-DOK3 in HEK-293T cells treated with 60 μmol/L SAB is shown. (O) The relative Protein levels of DOK3 in (N) ( n = 3). Data are shown as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Pull Down Assay, Incubation, Binding Assay, Kinetic Assay, Immunoprecipitation, Mutagenesis

Journal: Acta Pharmaceutica Sinica. B

Article Title: Augmentation of PRDX1–DOK3 interaction alleviates rheumatoid arthritis progression by suppressing plasma cell differentiation

doi: 10.1016/j.apsb.2025.06.006

Figure Lengend Snippet: Salvianolic acid B prevents the progression of collagen-induced arthritis by enhancing the interaction between PRDX1 and DOK3. (A) Clinical scores of arthritis in mice treated with vehicle, 1.5 mg/kg TF, and 15 mg/kg or 30 mg/kg SAB, and immunized with collagen in complete Freund's adjuvant ( n = 6). (B) Representative micro-CT images of the hind paws from mice treated with vehicle, TF, and SAB. (C) Joint destruction was graded based on the CT score in B ( n = 3). (D, E) histological score of the inflammation area in mice treated with vehicle, TF, and SAB ( n = 6) (D) and representative images of hematoxylin and eosin (H&E)-stained paw sections (E). Scale bar, 100 μm. (F) Serum titer of IgG2A analyzed from vehicle, TF, and SAB by ELISA ( n = 6). (G, H) The frequency of plasma cells (G) and GC B cells (H) in the spleen from mice treated with vehicle, TF, and SAB was analyzed by flow cytometry ( n = 6). (I) GSEA enrichment profiles of the B cell receptor pathway and CD40 pathway in the SAB-H treatment and model groups. (J) Heatmap depicting the expression levels of the B cell receptor signaling pathway and RA defined by the differentially downregulated genes in the SAB-H treatment group compared to the WT model group. (K) The biological process analysis of B cells for downregulated genes in the SAB-H treatment group compared to the model group. (L) Representative immunofluorescence staining images of PRDX1 (green) and DOK3 (red) in the spleen from the model and SAB treatment groups. Scale bar, 50 μm. (M) Representative immunofluorescence staining images of CD19 (green), P-JNK (red) in the spleen from the model and SAB treatment groups. Scale bar, 50 μm. (N) Relative fluorescence intensity of stained PRDX1 and DOK3 in the spleen from the model and SAB treatment groups ( n = 3). (O) Relative fluorescence intensity of stained CD19 and P-JNK in the spleen from the model and SAB treatment groups ( n = 3). Data are presented as mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, and ns indicates no significance.

Article Snippet: After in vitro stimulation of B cells, the Duolink assay was performed according to the manufacturer's instructions using Duolink Detection reagents Far Red (Sigma–Aldrich, St. Louis, MO, USA) and anti-PRDX1 rabbit (Proteintech, 15816-1-AP, Wuhan, China) and anti-DOK3 mouse primary antibodies (Santa Cruz Biotechnology, SC-390007, Santa Cruz, CA, USA).

Techniques: Adjuvant, Micro-CT, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Flow Cytometry, Expressing, Immunofluorescence, Fluorescence