rabbit anti dock8 antibody  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Identification of DOCK8 as marker of ai CD4 T cell and induction of SLE by DOCK8 + CD4 T cells (A) Expression of DOCK8 on CD45RB lo CD122 lo PD-1 + CD4 T cells. Membrane and cytosolic fractions of either whole T cells (Lanes 1 and 5), CD45RB hi CD122 hi CD4 T cells (Lanes 2 and 6), CD45RB lo CD122 lo PD-1 - CD4 T cells (Lanes 3 and 7), or CD45RB lo CD122 lo PD-1 + CD4 T cells (Lanes 4 and 8) from 12x OVA-immunized BALB/c mice, were subjected to 10% of polyacrylamide gel electrophoresis, transferred to Immobilon-P membrane, and stained with anti-DOCK8 Ab. (B) Increase of DOCK8 + CD4 T cells after repeated immunization with OVA without an adjuvant. Flow cytometry analyses of DOCK8 + CD4 T cell from splenic CD4 T cells of BALB/c mice after repeated 12x stimulation with PBS, 6x with OVA, or 12x with OVA. (C) DOCK8 + CD4 T cells transferred into recipient mice induced autoantibodies. Rheumatoid factor (RF), anti-Sm Ab, and anti-dsDNA Ab were quantified in the sera of BALB/c mice that had been pre-immunized 8x with OVA, depleted of CD4 T cells by anti-CD4 Ab, and then inoculated with DOCK8 − CD4 T cells or DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice. The control group was BALB/c mice pre-immunized 8x with OVA, CD4 T cell-depleted by anti-CD4 Ab, and inoculated with CD4 T cells from 12x PBS-immunized BALB/c mice. Anti-dsDNA Ab and anti-Sm Ab titers were represented by arbitrary unit (AU). Data were represented as mean ± SEM. Statistical assessment was by Student's t-test. ∗p<0.05. (D) Generation of CTL, i.e., IFNγ + CD8 T cells, after transfer of DOCK8 + CD4 T cells. BALB/c mice were immunized 12x with OVA, and CD4 T cells were isolated. Cells were transferred into the anti-CD4 Ab-treated recipient mice immunized 8x with OVA. Matured CTL, i.e., IFNγ + CD8 T cells, were measured 2 weeks after booster immunization 1x with OVA (n = 5). Data were represented as mean ± SEM. Statistical assessment was by Student's t-test. ∗p<0.05. (E) Skin lesion. a: Moderate dermatitis with dermal fibrosis, b: liquefaction degeneration (arrow) in the basal cells of epidermis, and c: panniculitis in the deep area of the dermis of mice pre-treated 8x with OVA, depleted of CD4 T cells, and inoculated with DOCK8 + T cells from 12x OVA-immunized mice. d: Discoid lupus-like advanced dermatitis observed in mice immunized 12x with OVA. e: Healed dermatitis with some fibrosis in 12x OVA-immunized mice treated with anti-DOCK8 Ab 24 h each before the 6x, 8x, 10x, and 12x OVA immunizations with OVA. H & E stain. (bar = 30μm; original magnification a, b, ×400; c, x300; d, e, x100). (F) Lesion other than kidney or skin. a: lung interstitial pneumonitis, b: interstitial pneumonitis accompanied by angiitis, c: liver pericholangitis, d: onion-skin lesion with amyloid deposition classical to lupus in the spleen, and f: thyroiditis, accompanied by giant cells, in the BALB/c mice 8x pre-immunized with OVA, CD4 T cell-depleted, and then inoculated with DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice. e: Classical onion-skin-like lesion with amyloid deposits in the spleen, and g: perineuritis observed in the dermis of the BALB/c mice immunized 12x with OVA. H & E stain. (bar = 30μm; original magnification ×200). See also .

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Marker, Expressing, Membrane, Polyacrylamide Gel Electrophoresis, Staining, Adjuvant, Flow Cytometry, Control, Isolation

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Generation of renal disease in recipient mice by the transfer of DOCK8 + CD4 T cells

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques:

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Summary of the lesions other than kidney in the BALB/c mice 8x pre-immunized with OVA, CD4 T cell-depleted, and inoculated with DOCK8 − CD4 T cells or DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Control

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: DOCK8 + CD4 T cell as T fh cell located at splenic red pulp, and its increase in peripheral blood of patients with SLE (A) a: Morphology of DOCK8 + CD4 T cell observed by immunoelectron microscopy, stained with rabbit anti-DOCK8 Ab and Au-tagged ant-rabbit IgG Ab. (bar = 1μm; original magnification ×12,000). b,c: Nuclear membrane portions of DOCK8 + CD4 T cell, stained likewise. (bar = 1μm; original magnification ×18,000; x24,000). (B) Markers of splenic DOCK8 + CD4 T cells. Flow cytometry for ICOS, CXCR5, and PD-1 expression in splenic DOCK8 + CD4 T cells taken 9 days after the final immunization of BALB/c mice immunized 12x with PBS versus OVA (Left). Flow cytometry analysis of ICOS, CXCR5, PD-1, GATA3, RORγt, T-bet, Bcl6, Ly6C, LFA1, FR4, and GL7 expression in splenic DOCK8 − CD4 T cells and DOCK8 + CD4 T cells isolated from 12x OVA-immunized BALB/c mice. (C) Cytokines released in culture supernatants of 1x10 6 /ml DOCK8 + or DOCK8 - CD4 T cells following incubation with 2 μg/mL of anti-CD3 and 5 μg/mL of anti-CD28 Ab at 37°C for 24hr. Data were represented as mean ± SEM. Statistical assessment was by Student's t-test. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.005, ∗∗∗∗p<0.001. (D) DOCK8 + CD4 T cells in the autopsied spleen of untreated patient with active SLE who died before starting therapy. Tissues were fixed in 10% formalin, deparaffinized by graded xylene, and graded alcohol sequentially, and serially treated with biotinylated anti-DOCK8 monoclonal Ab and rabbit anti-IFNγ Ab (for T cell staining), alkaline phosphatase avidin D, and anti-rabbit Novolink, followed by reaction with Fast Red (Red for DOCK8 + cells) and Histogreen (Blue for T cells). S: red pulp sinus. A; central artery. P: penicillar artery. (bar = 100μm; original magnification ×100). Inset; Magnified DOCK8 + CD4 T cells (Arrows). (bar = 20μm; original magnification ×400). (E) Circulating DOCK8 + CD4 T cells within CD4 T cells in the peripheral blood of patients with SLE and its relationship to the SLE disease activity index (SLEDAI). Data of control patients were depicted as; ■ rheumatoid arthritis, ▲ mixed connective tissue disease, △ polymyositis, ◆ systemic sclerosis, □ microscopic polyangiitis. Also shown was quantification of interferonα (IFNα) in sera of the same patients with SLE measured by ELISA. See also and .

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Immuno-Electron Microscopy, Staining, Membrane, Flow Cytometry, Expressing, Isolation, Incubation, Avidin-Biotin Assay, Activity Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: TCR revision in DOCK8 + CD4 T cells (A) Expression of V(D)J recombinase complex and related molecules in splenic CD4 T cells from 12x PBS-immunized BALB/c mice, and in DOCK8 − CD4 T cells and DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice. Two mice were analyzed for each group. (B) Western blot analysis of TCR signal transduction molecules in splenic CD4 T cells from 12x PBS-immunized BALB/c mice, and in DOCK8 − CD4 T cells and DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice. Three mice were analyzed for each group. GAPDH, control housekeeping glyceraldehyde 3-phosphate dehydrogenase gene. (C) TCR repertoire analysis. Heatmap visualization of TCR usage of a combination of TRV and TRJ genes in the splenic DOCK8 - CD4 T cells and DOCK8 + CD4 T cells from 12x OVA-immunized mice. See also and .

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Expressing, Western Blot, Transduction, Control

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Autoantibody microarray study Autoantibodies produced in BALB/c mice pre-immunized 8x with OVA, depleted of CD4 T cells, and inoculated with DOCK8 − CD4 T cells or DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice. Control was autoantibodies produced in BALB/c mice pre-immunized 8x with OVA, depleted of CD4 T cells, and inoculated with CD4 T cells from 12x PBS-immunized BALB/c mice. Two mice were analyzed for each group.

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Microarray, Produced, Control

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Expression of transcriptional regulators in DOCK8 + CD4 T cells (A) Transcriptional regulators responsible for TCR revision. Western blot analysis of DOCK8 and transcriptional regulators in splenic CD4 T cells from 12x PBS-immunized BALB/c mice, and in DOCK8 − CD4 T cells and DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice. Three mice were analyzed for each group. (B) Upregulated genes in DOCK8 + CD4 T cells. Results of gene expression microarray analysis, abstract from ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159240 ). Ratio of mRNA expression of splenic DOCK8 + CD4 T cells from 12x OVA-immunized BALB/c mice in comparison with splenic DOCK8 − CD4 T cells from 12x OVA-immunized mice, given as a log 10 scale, wherein 2, 3, 4, 5, and 6 indicated x10 2 , x10 3 , x10 4 , x10 5 , and x10 6 , respectively.

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Expressing, Western Blot, Gene Expression, Microarray, Comparison

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Enhanced chromatin accessibility in DOCK8 + CD4 T cells (A) Chromatin-immunoprecipitation sequencing (ChIP-seq) assay for V(D)J chromatin accessibility. Left panel: Histone acetylation (AcH3) and H3K4 methylation (H3K4me3) of the TCRA gene of splenic ai CD4 T cells from 8x SEB-stimulated BALB/c mice. Splenic CD4 T cells of 8x SEB-stimulated BALB/c mice which produced amply high titers of autoantibodies were assayed. TEA, TCRα promoter. Eα, TCRα enhancer. MyoD, myoblast determination 1 as negative controls. Right panel: Histone acetylation (AcH3) and H3K4 methylation (H3K4me3) of the TCRB gene of splenic ai CD4 T cells from 8x SEB-stimulated BALB/c mice as above. Eβ, TCRβ enhancer. (B) TCRα chain revision in the splenic CD4 T cells from the mice immunized 8x with SEB producing high titers of autoantibodies was determined by LM-PCR detection of dsDNA breaks at the RSS flanking the TCRAJ12. WT, wild-type.

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: ChIP-sequencing, Methylation, Produced

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Treatment with anti-DOCK8 Ab (A) Effect of anti-DOCK8 mAb treatment on autoantibody production in12x OVA-immunized mice. Autoantibodies, rheumatoid factor (RF), anti-Sm Ab, and anti-dsDNA Ab, were quantified in the sera of 12x PBS-immunized or 12x OVA-immunized mice: Mice received anti-DOCK8 Ab (100μg) or control rabbit IgG (100μg) 24 h each before the 6x, 8x, 10x, and 12x immunizations with OVA. Anti-Sm Ab and anti-dsDNA Ab titers were represented by arbitrary unit (AU). Data were represented as mean ± SEM. Statistical analysis was by Student's t-test; ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.005. (B) Effects of anti-DOCK8 Ab treatment on renal disease. Proteinuria measured 9 days after the final immunization in 12x PBS-immunized or 12x OVA-immunized BALB/c mice, graded with a score of 0 (<30 mg/dL); 1 (30–99 mg/dL); 2 (100–299 mg/dL); or 3 (300–999 mg/dL). (C) Effects of anti-DOCK8 mAb treatment on autoantibody production and renal disease in (NZBxNZW) F1 female mice. Autoantibodies, anti-dsDNA Ab, anti-Sm Ab, and rheumatoid factor (RF), and proteinuria quantified by ELISA in (NZBxNZW) F1 female mice, which were treated either with anti-DOCK8 mAb (100 μg) or control rabbit IgG (100 μg) every week from 15 to 24 weeks (arrow). Data were represented as mean ± SEM. Statistical assessment performed by Student's t-test; ∗p<0.05, ∗∗p<0.01, ∗∗∗∗p<0.001.

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet: Renal lesion of BALB/c mice immunized 12x with either PBS or OVA, and treated either with anti-DOCK8 Ab (100μg) or control rabbit IgG (100μg) 24 h each before the 6x, 8x, 10x, and 12x OVA immunizations with OVA

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Control

Journal: iScience

Article Title: DOCK8-expressing T follicular helper cells newly generated beyond self-organized criticality cause systemic lupus erythematosus

doi: 10.1016/j.isci.2021.103537

Figure Lengend Snippet:

Article Snippet: Anti-DOCK8 rabbit Ab , Proteintech , Cat#11622-1-AP RRID; AB_10216360.

Techniques: Activation Assay, Recombinant, Avidin-Biotin Assay, Plasmid Preparation, Extraction, Membrane, Staining, Diagnostic Assay, Magnetic Beads, Protease Inhibitor, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Isolation, Purification, Gene Expression, Microarray

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Interacting proteins of septin 7 in PBL of healthy controls.

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques:

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Septin 7 interacted with DOCK8 in equine PBL. Representative western blots of immunoprecipitations of septin 7 (A) and DOCK8 (B) . DOCK8 was detected at 190 kDA in input lysates of PBL (input PBL) as well as after immunoprecipitation of septin 7 (A) . Septin 7 was detected at 49 kDA in input lysates of PBL (input PBL) as well as after immunoprecipitation of DOCK8, verifying an interaction between these two proteins. In respective isotype controls (rabbit IgG; rat IgG 2c), blank (IP buffer) and negative control MALT1; neither septin 7 nor DOCK8 was detected (C) .

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques: Western Blot, Immunoprecipitation, Negative Control

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Characterization of DOCK8 expression in horse lymphocytes. (A – C) Representative DOCK8 expression in different lymphocyte subsets. Flow cytometry analyses demonstrate that DOCK8 is expressed in CD4 + T cells, CD8 + T cells and in B cells (white histograms) with high intensity. White histograms represent specific stainings, grey histograms represent respective isotype controls. Y-axis show the amount of cells included in measurement; x-axis show the intensity of DOCK8 staining. (D) Statistical analyses of geo mean fluorescence intensity of DOCK8 expression in lymphocyte subsets of 12 healthy controls. While there was no difference between DOCK8 expression intensity in CD4 + T cells (white column) and CD8 + T cells (light grey column), DOCK8 was significantly higher (*p ≤ 0.05) expressed in B cells (dark grey column) compared to T cells.

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Diminished DOCK8 protein levels in PBL of ERU cases. (A) Representative signal abundances of DOCK8 in PBL of controls (left panel) and ERU (right panel) detected by western blot. DOCK8 signal abundance in PBL of diseased specimen was decreased compared to controls. Lower molecular extra bands result from further reactivities of the polyclonal rabbit anti DOCK8 antibody. (B) Compared to healthy controls (n = 16), DOCK8 levels in ERU PBL (n = 36) were significantly (***p ≤ 0.001) decreased to 63% of physiological expression rate (set to a 100%). All protein abundances of DOCK8 were normalized to beta actin. (C) Analyzes of lymphocyte subset composition in healthy controls and autoimmune cases revealed no significant differences. (D) Statistical analysis of DOCK8 expression differences in lymphocyte subsets of healthy controls (white bars; n = 13) and ERU cases (grey bars; n = 12). DOCK8 expression intensity (geo MFI) was decreased in all lymphocyte subsets of ERU cases. In CD8 + T cells, no considerable DOCK8 expression difference between control PBL and ERU cases could be detected, whereas in CD4 + T cells of ERU horses, geo MFI was reduced to 85% compared to healthy controls. In B cells of autoimmune cases, a significant decrease of DOCK8 geo MFI to 57% was detected (*p ≤ 0.05).

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques: Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Overrepresented signaling pathways of DOCK8 interacting proteins in PBL of healthy controls.

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques: Protein-Protein interactions

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: Overrepresented signaling pathways of DOCK8 interacting proteins in PBL of ERU cases.

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques: Protein-Protein interactions

Journal: Scientific Reports

Article Title: Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

doi: 10.1038/s41598-018-30753-7

Figure Lengend Snippet: DOCK8 interacted with ILK in equine PBL. Representative western blots of immunoprecipitations of DOCK8 (A) and ILK (B) . ILK was detected at 59 kDA in input lysates of PBL (input PBL) as well as after immunoprecipitation of DOCK8 (A) . DOCK8 was detected at 190 kDA in input lysates of PBL (input PBL) as well as after immunoprecipitation of ILK (B) , verifying an interaction between these two proteins. In respective isotype controls (rb IgG; mIgG) and blank (IP buffer), neither ILK nor DOCK8 was detected.

Article Snippet: 40 μl of Protein G Sepharose Beads (GE Healthcare, Freiburg, Germany) per 1 mg protein were washed with IP-buffer diluted with TBS and incubated separately with rat anti-septin 7 antibody (clone 19A4, self-made monoclonal rat anti-septin 7 antibody, isotype IgG2c; neat), rabbit polyclonal anti-DOCK8 antibody (H-159, Santa Cruz, Heidelberg, Germany; 6 μg/ml) diluted in IP-buffer or respective isotype controls were used.

Techniques: Western Blot, Immunoprecipitation

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A and B) Proliferation measured by Cell Trace Violet dilution (A) and IL-2 secretion in culture supernatants (B) by CD4+CD25– Teffs isolated from the spleens of Dock8–/– and WT mice cultured for 3 days with anti-CD3+anti-CD28–coated beads. (C) Percentage of CD25+FOXP3+ Tregs among CD4+ cells in the thymuses, spleens, and LNs of Dock8–/– and WT mice. n = 17 mice from each group for the thymus, n = 31 mice from each group for the spleen, n = 7 mice from each group for the LN. (D) Percentages of CD44–CD62Lhi rTregs and CD44+CD62Llo aTregs of total CD4+FOXP3+ cells in the spleens of Dock8–/– and WT mice. n = 5 mice from each group. (E) Representative FACS plots of intracellular FOXP3 and CTLA-4 and surface CD25 expression gating on CD4+FOXP3+ splenocytes (left). Quantitative analysis of surface CD25 expression by splenic CD4+FOXP3+ cells from Dock8–/– mice and WT controls. n = 29 mice from each group. (F) qPCR analysis of Foxp3 and Il2ra mRNA levels in FACS-sorted CD4+CD25+CD39+ Tregs from Dock8–/– and WT mice. Results are expressed as fold increase relative to the WT control ratio of the mRNA of interest/b2microglobulin. (G) Suppression of the proliferation of CD4+CD25– Teffs by CD4+CD25+CD39+ Tregs from Dock8–/– mice and WT controls. Teff proliferation was measured by FACS analysis of Cell Trace Violet dilution. The left panel is a representative experiment; the right panel shows the pooled results. (H) qPCR analysis of Tgfb and Il10 mRNA expression by sorted CD4+CD25+CD39+ Tregs from Dock8–/– mice and WT controls. Symbols represent individual mice, and error bars represent mean and SEM. Results in A, B, and F–H are representative of 3 independent experiments. t test, NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell lysates were incubated with anti-DOCK8 (rabbit polyclonal, Sigma-Aldrich) or anti-STAT5 (C-17, Santa Cruz).

Techniques: Isolation, Cell Culture, Expressing

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A) Representative FACS analysis of pSTAT5 staining (left) and percentage of pSTAT5+ cells (right) in splenic CD4+YFP+ Tregs from Foxp3YFP–Cre/Dock8flox/flox mice and Foxp3YFP–Cre controls stimulated for 15 minutes with 10 ng/ml mouse IL-2. (B) Relative pSTAT5 content in CD4+FOXP3+YFP– and CD4+FOXP3+YFP+ Tregs from Foxp3YFP–Cre/+/Dock8flox/flox female mice and Foxp3YFP–Cre/+ controls at baseline and following stimulation with increasing concentrations of IL-2 for 15 minutes. The graph plots the relative pSTAT5/STAT5 MFI ratio normalized to the Foxp3YFP–Cre/+ YFP– Treg baseline ratio. (C) Representative immunoblot of STAT5, pSTAT5, and DOCK8 in DOCK8 immunoprecipitates and cell lysates from WT T cells, and quantification of 4 individual experiments, showing the ratio of immunoprecipitated STAT5/DOCK8. Results were normalized to media alone samples. (D) Representative immunoblot of STAT5, pSTAT5, and DOCK8 in STAT5 immunoprecipitates and cell lysates from WT T cells, and quantification of 3 individual experiments, showing the ratio of immunoprecipitated DOCK8/STAT5. Results were normalized to media-alone samples. Results in A and B are representative of 3 independent experiments using 2–3 mice per group in each. Error bars represent the mean and SEM. Significance was determined by unpaired t test in A, C, and D, while ANOVA was used to compare the curves in B. ns P > 0.05, ***P < 0.001.

Article Snippet: Cell lysates were incubated with anti-DOCK8 (rabbit polyclonal, Sigma-Aldrich) or anti-STAT5 (C-17, Santa Cruz).

Techniques: Staining, Western Blot, Immunoprecipitation

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A) Baseline F-actin content of CD4+CD25+ Tregs from Dock8–/– and WT mice, and effect of CD3 crosslinking on the F-actin content of Tregs from Dock8–/– and WT mice. Results are expressed as the change in the MFI of F-actin from the baseline (time 0). (B) Representative images of CD4+CD25+ Tregs from Dock8–/– mice and WT controls plated on anti-CD3– and ICAM-1–coated glass chambers and stained for F-actin by phalloidin at 5 and 45 minutes (original magnification, ×100). (C) Relative number of adherent cells per unit area at 5 minutes. (D) Quantitative analysis of F-actin staining of Tregs after 5 and 45 minutes of stimulation. (E) Localization of pTYR, F-actin, and DOCK8 in Dock8–/– and WT Tregs at 10 minutes (original magnification, ×100). (F) A representative TIRF image of DOCK8 and actin distribution across the synapse after 10 minutes of synapse formation. The graph shows line scan profiles of DOCK8 and ACTIN across the middle of the cell; the green trace represents ACTIN intensity distribution, and the red trace represents DOCK8 intensity distribution. Note that DOCK8 and ACTIN intensities coincide in the cell periphery, represented by the peaks at the beginning and end of the line scan profiles. (G) Side view of 2 representative T cells, showing the whole cell ACTIN, pTYR, and DOCK8 distribution. Cells stained with DOCK8, pTYR, and ACTIN were imaged using confocal microscopy, and the images show a side view of maximum intensity projection of the confocal images. (H) Quantification of cell edge roughness determined by “shape descriptors” utility of ImageJ at 5 and 45 minutes. (I) Measurement of immune synapse instability, as denoted by the kinapse index (transient interactions index > 1) at 5 and 45 minutes. (J) Relative staining intensity of pTYR, phospho-ZAP70 (pZAP70), and TALIN at 10 minutes. (K) Transendocytosis of CD86-GFP by Tregs from Dock8–/– and WT mice cocultured with CD86-GFP–expressing CHO cells. The percentage of GFP+ Tregs of total Tregs is shown. Results in A and K are representative of 3 independent experiments. Results in B–J are representative of 2 independent experiments. Symbols represent individual measurements, and error bars represent mean and SEM. Significance was determined by unpaired t test. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cell lysates were incubated with anti-DOCK8 (rabbit polyclonal, Sigma-Aldrich) or anti-STAT5 (C-17, Santa Cruz).

Techniques: Staining, Confocal Microscopy, Expressing

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A and B) CD4+CD25+CD39+YFP+ (DOCK8-deficient) and YFP– (DOCK8-sufficient) Tregs were sorted from Foxp3YFP–Cre/+/Dock8flox/flox female mice. Tregs were cultured overnight in media alone (A) or with anti-CD3+CD28 beads (B). Heatmaps of selected genes differentially expressed in YFP+ and YFP– Tregs from Foxp3YFP–Cre/+Dock8flox/flox female mice. The cutoff for significance was P < 0.05. P values were calculated using the Wald test for differential expression analysis. P values were corrected afterward for multiple testing. Expression of genes is centered and scaled by row to highlight differences in each gene sample. Each column represents an individual mouse. (C and D) CD4+CD25+CD39+ Tregs were FACS sorted from Dock8–/– and WT mice. RNA was prepared from Tregs directly after isolation (C) or after 24-hour culture with anti-CD3+CD28 beads (C and D). qPCR results are expressed as fold increase of mRNA of interest/b2microglobulin ratio relative to the unstimulated WT Tregs. (E) MFI of surface marker staining on YFP– DOCK8-sufficient and YFP+ DOCK8-deficient CD4+CD25+CD39+ Tregs from Foxp3YFP–Cre/+/Dock8flox/flox female mice. Symbols represent individual mice. Bars in C–E represent the mean and SEM. t test, NS P > 0.05, *P < 0.05, ***P < 0.001.

Article Snippet: Cell lysates were incubated with anti-DOCK8 (rabbit polyclonal, Sigma-Aldrich) or anti-STAT5 (C-17, Santa Cruz).

Techniques: Cell Culture, Expressing, Isolation, Marker, Staining