Journal: Nature Communications

Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

doi: 10.1038/s41467-020-16471-7

Figure Lengend Snippet: a , b , e, f, h Representative images of Jurkat T cells allowed to adhere on poly-L-lysine slides. Bars represent 5-μm scale. Jurkat T cells were fixed and stained for CD3ε or CD3ζ (green) and CNX (red) ( a ), CD3ζ (green) and EEA1 or LAMP1 or IRAP or Stx6 (red) ( b ). c Quantification of early and late endosomal marker colocalization with CD3ζ ( n = 13 cells for EEA1 and IRAP; n = 11 for LAMP1 and Stx6). d Immunoblot analysis of IRAP, Lck and CD3ζ after immunoprecipitation with anti-IRAP or an isotype control in Jurkat T cells. e , f CD3ζ colocalization with Stx6 ( n = 10 cells, **** p < 0.0001) ( e ) and Rab4 ( n = 10 cells, *** p = 0.0001) ( f ) in wt or IRAP ko Jurkat T cells transduced with a CD3ζ-GFP reporter ( e ) or a Rab4-GFP reporter ( f ). Bars represent 5-μm scale. g CD3ζ expression on the cell surface of wt or IRAP ko Jurkat T cells expressing a CD8-CD3ζ reporter, measured by flow cytometry ( n = 6 experiments, *** p = 0.0008). h CD3ζ (red) colocalization with IRAP (green) in wt or DnM2-deficient Jurkat T cells. Bars represent 5 μm scale. i CD3ε expression on the cell surface of wt or DnM2-deficient Jurkat T cells, measured by flow cytometry ( n = 3 experiments, ** p = 0.0017). j CD3ε expression on the cell surface of wt or IRAP ko Jurkat T cells, measured by flow cytometry ( n = 6 experiments, * p = 0.0340). k CD3ε expression on the cell surface of wt or AP2μ-deficient Jurkat T cells, measured by flow cytometry ( n = 2 experiments). All p -values ( e , f , g , i , j ) were calculated with two-tailed unpaired t tests. Each symbol represents an individual cell ( c , e , f ) or an independent experiment ( g , i , j ). Values represent mean ± SEM. For additional information, see Supplementary Fig. .

Article Snippet: For detection by immunoblot, the following antibodies were used: rabbit anti-IRAP (provided by S. Keller, Virginia University), rabbit anti-IRAP (D7C5, Cell Signaling Technology), mouse anti-IRAP (3E1, Cell Signaling Technology), mouse anti-Lck (3A5, Santa Cruz Biotechnology), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), mouse anti-β-actin (AC-15, Sigma-Aldrich), rabbit anti-LAT (Millipore), mouse anti-CD247 (pY142) (K25-407.69, BD Pharmingen), rabbit anti-pLAT, rabbit anti-ZAP-70 (D1C10E), rabbit anti-pZAP70 (65E4), rabbit anti-pPLC γ 1, rabbit anti-PLC γ 1 and rabbit anti-pSrc (D49G4) (all from Cell Signaling Technology).

Techniques: Staining, Marker, Western Blot, Immunoprecipitation, Transduction, Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

doi: 10.1038/s41467-020-16471-7

Figure Lengend Snippet: a Immunoblot analysis of TCR signalling molecules in wt and IRAP ko Jurkat T cells after activation by anti-CD3ε/CD28 for various time points. Values represent mean ± SEM of pool of three independent experiments for all signalling components excepting pPLCγ ( n = 4) and pZAP70 ( n = 2). pLAT * p = 0.0429, pCD3ζ * p = 0.0231, pLck * p = 0.0107, pPLCγ * p 1.5 min = 0.0262, * p 5 min = 0.0199. b , c IL-2 response of Jurkat T cells measured by ELISA: b wt or IRAP ko Jurkat T cells were incubated for 6 h with Raji B cells presenting the SEE superantigen, c wt or IRAP ko Jurkat T cells expressing the MART1 TCR were incubated overnight with Daju-A2 cells presenting the MART1 peptide. Values represent mean ± SEM of three replicates of one representative experiment of three independent experiments. SEE **** p < 0.0001, *** p = 0.0004, MART1 **** p < 0.0001, *** p = 0.0002. d Quantification of TIRF microscopy of IRAP, Rab4, Lck, pZAP70 and LAT membrane recruitment in CD3ε/CD28-activated Jurkat T cells. Wt or IRAP ko Jurkat T cells were activated (CD3/CD28) or not (Ctrl) for 10 min on CD3ε/CD28-coated slides, fixed and stained for IRAP or pZAP70 or LAT. For Rab4 and Lck, cells were transfected with Rab4-GFP and Lck-GFP, respectively. Each dot represents a cell (IRAP: wtCtrl n = 28, wtCD3/CD28 n = 34, *** p = 0.0005; Rab4: wtCtrl n = 30, wtCD3/CD28 n = 46, IRAPkoCtrl n = 30, IRAPkoCD3/CD28 n = 45, * p = 0.016, *** p = 0.0001; Lck wtCtrl n = 23, wtCD3/CD28 n = 41, IRAPkoCtrl n = 21, IRAPkoCD3/CD28 n = 42, * p = 0.033; pZAP70 wtCtrl n = 33, wtCD3/CD28 n = 35, IRAPkoCtrl n = 31, IRAPkoCD3/CD28 n = 34, ** p = 0.009, *** p = 0.0001; LAT wtCtrl n = 8, wtCD3/CD28 n = 19, IRAPkoCtrl n = 9, IRAPkoCD3/CD28 n = 14). Values represent mean ± SEM. All p -values ( a , b , c , d ) were calculated with two-tailed unpaired t tests. e Confocal microscopy of SEE-pulsed Raji B cell/Jurkat T-cell conjugates. Cells were stained for: Lat (red), Lck (green)—right panel, Lat (red), CD3ε (green)—middle panel and pZAP70 (red), IRAP (green)—left panel. Bars represent 5-μm scale. Images are representative of three independent experiments. For additional information, see Supplementary Fig. .

Article Snippet: For detection by immunoblot, the following antibodies were used: rabbit anti-IRAP (provided by S. Keller, Virginia University), rabbit anti-IRAP (D7C5, Cell Signaling Technology), mouse anti-IRAP (3E1, Cell Signaling Technology), mouse anti-Lck (3A5, Santa Cruz Biotechnology), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), mouse anti-β-actin (AC-15, Sigma-Aldrich), rabbit anti-LAT (Millipore), mouse anti-CD247 (pY142) (K25-407.69, BD Pharmingen), rabbit anti-pLAT, rabbit anti-ZAP-70 (D1C10E), rabbit anti-pZAP70 (65E4), rabbit anti-pPLC γ 1, rabbit anti-PLC γ 1 and rabbit anti-pSrc (D49G4) (all from Cell Signaling Technology).

Techniques: Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Microscopy, Staining, Transfection, Two Tailed Test, Confocal Microscopy

Journal: Nature Communications

Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

doi: 10.1038/s41467-020-16471-7

Figure Lengend Snippet: a CD3ζ reporter composed of a CD3ζ chain followed by a GFP molecule, an mCherry molecule and an SH2 domain of ZAP-70. At the activated state, Lck phosphorylates the CD3ζ ITAMs, which then recruit the SH2 domain of ZAP-70. Then GFP and mCherry come to a distance where FRET can occur. b Representative images and quantification of GFP average lifetime in CD3ε/CD28-activated Jurkat T cells. Jurkat T cells expressing the CD3ζ reporter and transduced with either shNT or shIRAP lentivirus were activated on CD3ε/CD28-coated slides for 10 min, fixed and GFP average lifetime was measured by FRET–FLIM. Green symbols represent quantification at the plasma membrane (mb), and red symbols quantification in endosomes (endo). Each dot represents an individual cell (GFP n = 46; GFP-mCherry n = 38; wt mb n = 43; ko mb n = 39; wt endo n = 28; ko endo n = 30, ** p = 0.0064, **** p < 0.0001). Bars represent 5-μm scale . c Wt or IRAP ko Jurkat T cells were activated on CD3ε/CD28-coated slides for 10 min and stained with anti- CD3ζ pY142 and anti-Stx6 antibodies. Bars represent 5-μm scale . d , e Colocalization of Stx6 (red) with Lck (green) in wt or IRAP ko Jurkat T cells transfected with Lck(Y505F)-GFP ( d , n = 8 wt, n = 8 IRAP ko cells, ** p = 0.0070) or Lck-GFP ( e , n = 8 wt, n = 8 IRAP ko cells, * p = 0.0486). Bars represent 5-μm scale . f Duolink proximity ligation assay of IRAP with various signalling proteins on conjugates formed by Raji B cells presenting SEE superantigen to wt Jurkat T cells expressing Rab4-GFP. Interactions of less than 40-nm distance between the two proteins appear as a red dot. Zoom, original magnification × 4. Bars represent 5 μm scale. At least 20 cells per condition were quantified. Lck ** p = 0.0027, CD3ε *** p = 0.0008, ZAP *** p = 0.0001, LAT ** p = 0.0054. Each dot represents an individual cell, and all values represent mean ± SEM ( b , d, e, f ). All p -values were calculated with two-tailed unpaired t tests. For additional information, see Supplementary Fig. .

Article Snippet: For detection by immunoblot, the following antibodies were used: rabbit anti-IRAP (provided by S. Keller, Virginia University), rabbit anti-IRAP (D7C5, Cell Signaling Technology), mouse anti-IRAP (3E1, Cell Signaling Technology), mouse anti-Lck (3A5, Santa Cruz Biotechnology), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), mouse anti-β-actin (AC-15, Sigma-Aldrich), rabbit anti-LAT (Millipore), mouse anti-CD247 (pY142) (K25-407.69, BD Pharmingen), rabbit anti-pLAT, rabbit anti-ZAP-70 (D1C10E), rabbit anti-pZAP70 (65E4), rabbit anti-pPLC γ 1, rabbit anti-PLC γ 1 and rabbit anti-pSrc (D49G4) (all from Cell Signaling Technology).

Techniques: Expressing, Transduction, Staining, Transfection, Proximity Ligation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

doi: 10.1038/s41467-020-16471-7

Figure Lengend Snippet: a Intracellular IRAP expression in wt, IRAP ko and IRAP ko cells reconstituted with IRAP E465A or IRAP 3CA, measured by flow cytometry ( n = 3 experiments, ** p wt = 0.0034, ** p E465A = 0.0071, *** p = 0.0002). Values represent mean ± SEM of three independent experiments. b Representative images of Jurkat T cells expressing endogenous IRAP (wt) and IRAP ko cells expressing IRAP E465A or IRAP 3CA via lentiviral transductions. The cells were activated on CD3ε/CD28-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and Stx6. Bars represent 5-μm scale. c CD3ε expression on the cell surface of wt, IRAP ko Jurkat T cells and IRAP ko reconstituted with IRAP E465A and IRAP 3CA, measured by flow cytometry ( n = 3 experiments, *** p = 0.0006, * p = 0.0398). Values represent mean ± SEM of three independent experiments. d Representative images of Jurkat T cells expressing endogenous IRAP (wt) and IRAP ko cells expressing IRAP E465A or IRAP 3CA via lentiviral transductions. The cells were activated on CD3ε/CD28-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and phospho-CD3ζ. Bars represent 5-μm scale. e wt, IRAP ko Jurkat T cells and IRAP ko transduced with lentiviruses expressing IRAP E465A or IRAP 3CA were incubated with SEE-pulsed Raji cells. Values represent mean ± SEM of three replicates of one representative experiment of three independent experiments. Wt: * p = 0.0116, ** p = 0.0010, **** p < 0.0001, IRAP E465A: ** p 0.01 = 0.0037, *** p = 0.0001, **** p < 0.0001, ** p 100 = 0.0030. All p -values ( a , c , e ) were calculated with two-tailed unpaired t tests. f Immunoblot analysis of IRAP and CD3ζ after immunoprecipitation with anti-IRAP in IRAP ko Jurkat T cells reconstituted with IRAP E465A or IRAP 3CA. Images are representative of two independent experiments.

Article Snippet: For detection by immunoblot, the following antibodies were used: rabbit anti-IRAP (provided by S. Keller, Virginia University), rabbit anti-IRAP (D7C5, Cell Signaling Technology), mouse anti-IRAP (3E1, Cell Signaling Technology), mouse anti-Lck (3A5, Santa Cruz Biotechnology), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), mouse anti-β-actin (AC-15, Sigma-Aldrich), rabbit anti-LAT (Millipore), mouse anti-CD247 (pY142) (K25-407.69, BD Pharmingen), rabbit anti-pLAT, rabbit anti-ZAP-70 (D1C10E), rabbit anti-pZAP70 (65E4), rabbit anti-pPLC γ 1, rabbit anti-PLC γ 1 and rabbit anti-pSrc (D49G4) (all from Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Staining, Transduction, Incubation, Two Tailed Test, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

doi: 10.1038/s41467-020-16471-7

Figure Lengend Snippet: a OT1 effector T cells were spread on poly-lysine (nonstim.) or CD3ε-coated (stim.) slides for 10 min, fixed and stained with antibodies specific for IRAP and Stx6. Each dot represents a cell ( n = 11 cells per condition). Bars represent 5-μm scale. b OT1 T cells were spread on CD3ε-coated slides for 10 min, fixed and stained with antibodies specific for IRAP and the ζ chain ( n = 8 cells per condition). Bars represent 5-μm scale. Images are representative of two independent experiments. c Confocal microscopy and tri-dimensional reconstitution of conjugates formed by SIINFEKL-pulsed dendritic cells and OT1 naive T cells. After fixation, the cells were stained with specific antibodies for LFA-1 (red), IRAP (green) and DAPI (grey). Images are representatives for two independent experiments. Bars represent 5-μm scale. d Confocal microscopy of conjugates formed by SIINFEKL-pulsed dendritic cells and OT1 naive T cells. After fixation, the cells were stained with specific antibodies for IRAP (green) and TCRb (red). Bars represent 5-μm scale. Images are representatives for two independent experiments.

Article Snippet: For detection by immunoblot, the following antibodies were used: rabbit anti-IRAP (provided by S. Keller, Virginia University), rabbit anti-IRAP (D7C5, Cell Signaling Technology), mouse anti-IRAP (3E1, Cell Signaling Technology), mouse anti-Lck (3A5, Santa Cruz Biotechnology), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), mouse anti-β-actin (AC-15, Sigma-Aldrich), rabbit anti-LAT (Millipore), mouse anti-CD247 (pY142) (K25-407.69, BD Pharmingen), rabbit anti-pLAT, rabbit anti-ZAP-70 (D1C10E), rabbit anti-pZAP70 (65E4), rabbit anti-pPLC γ 1, rabbit anti-PLC γ 1 and rabbit anti-pSrc (D49G4) (all from Cell Signaling Technology).

Techniques: Staining, Confocal Microscopy

Journal: Nature Communications

Article Title: IRAP-dependent endosomal T cell receptor signalling is essential for T cell responses

doi: 10.1038/s41467-020-16471-7

Figure Lengend Snippet: a TCRb expression on the cell surface of effector OT1 or IRO T cells (culture day 7), measured by flow cytometry. Each symbol represents an individual experiment. Values represent mean ± SEM of three independent experiments, * p = 0.0468. b Immunoblot analysis of effector OT1 or IRO T cells after activation for the indicated time points by anti-CD3ε/CD28 antibody. Values on quantification graphs represent mean ± SEM of pool of three independent experiments, * p = 0.0352, ** p = 0.0031. c CD8 + T-cell absolute number quantification in OT1 or IRO mouse lymph nodes by flow cytometry. Values represent mean ± SEM of five mice from two independent experiments, * p = 0.0172. CD8 + cells are gated on CD45 + TCRb + live cells. d Quantification of number of OT1 and IRO T cells after activation by DC2.4 loaded with N4 or Q4 peptides. Values represent mean ± SEM of three or four independent experiments, Q4 day 3 * p = 0.017, Q4 day 7 * p = 0.011. Representative proliferation graphs at day 3 are shown. e CD8 + and CD4 + T-cell absolute number quantification in wt (IRAP loxlox ) or IRAP Tcellko mouse lymph nodes or spleen by flow cytometry. CD8 + and CD4 + cells are gated on CD45 + TCRb + live cells. Values represent mean ± SEM of six mice from two independent experiments, LN CD8 + *** p = 0.0003, CD4 + ** p = 0.0017; spleen CD8 + ** p = 0.0037, CD4 + ** p = 0.0096. All p -values ( a , b , c , d , e ) were calculated with two-tailed unpaired t tests. For additional information, see Supplementary Figs. 4, 5, 6 and 7.

Article Snippet: For detection by immunoblot, the following antibodies were used: rabbit anti-IRAP (provided by S. Keller, Virginia University), rabbit anti-IRAP (D7C5, Cell Signaling Technology), mouse anti-IRAP (3E1, Cell Signaling Technology), mouse anti-Lck (3A5, Santa Cruz Biotechnology), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), mouse anti-β-actin (AC-15, Sigma-Aldrich), rabbit anti-LAT (Millipore), mouse anti-CD247 (pY142) (K25-407.69, BD Pharmingen), rabbit anti-pLAT, rabbit anti-ZAP-70 (D1C10E), rabbit anti-pZAP70 (65E4), rabbit anti-pPLC γ 1, rabbit anti-PLC γ 1 and rabbit anti-pSrc (D49G4) (all from Cell Signaling Technology).

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

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: IRAP-deficient mice show less severe IgE- and IgG-induced anaphylactic reactions and experimental arthritis. (A) 24h after sensitization with anti-DNP IgE wild-type ( WT) and IRAP-deficient (IRAP KO ) mice were challenged with antigen (DNP-HSA) to induce passive systemic anaphylaxis (PSA). The drop in body temperature was evaluated. Data presented are the mean ± s.e.m. with 9 mice (pooled from 3 experiments). (B) Released serum MCPT-1 chymase collected at the end of temperature measurements was evaluated in the two groups of mice as well as in unsensitized control mice. (C) WT FcγRIIA Tg and FcγRIIA Tg IRAP KO mice were immunized with rabbit IgG and 7 days later mice were challenged with rabbit IgG to induce active systemic anaphylaxis (ASA). The drop in body temperature was evaluated. Data are presented as mean ± s.e.m. with 6 mice (pooled from 2 experiments). (D) ASA was monitored by evaluating plasma platelet counts collected at the end of temperature measurements in the two groups of mice as well as in non-immunized control mice. Data are the mean ± s.e.m. (E) Serum MCPT-1 chymase collected at the end of temperature measurements was measured in the two groups of mice as well as in non-immunized control mice. Data are the mean ± s.e.m. (F) WT and IRAP KO or WT FcγRIIA Tg and FcγRIIA Tg IRAP KO mice were injected with an anti-collagen type II Ab cocktail (day 0) followed by injection of LPS (day 4). Photographs shows representative hematoxylin/eosin (HE) staining of ankle sections as well as the macroscopic appearance of hind legs for each genotype at day 8. (G) Arthritis development was monitored by measuring paw thickness starting 5 days after injection of the Ab cocktail. (H) Arthritis scores were also evaluated according to the provided scoring system (Chondrex). Data are the mean ± s.e.m. from 4 to 7 mice/group. Statistical analysis was done using the two-way ANOVA followed by Sidak’s post-hoc test (A, C, G, H) or the unpaired Student’s t test (B, D, E) . *: P < 0.05; **: P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Control, Clinical Proteomics, Injection, Staining

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: IRAP-deficient mice exhibit a diminished degranulation response and cytokine/chemokine production in BMMCs and PDMCs. WT and IRAP-deficient (IRAP KO ) BMMCs (A, C) and PDMCs (B, D) were sensitized with anti-DNP IgE. (A, B) Degranulation was monitored by flow cytometry determining CD63 expression (% expression is indicated) as a surrogate marker of mast cell degranulation 10 min after addition of PBS (basal expression) or antigen (30 ng/mL of DNP-HSA). A representative experiment (left panel) and the quantitative analysis of indicated number of experiments are shown (right panel). (C, D) Degranulation was also determined by measuring the net release of the granular enzyme β-hexosaminidase in BMMCs and PDMCs after stimulation with IgE/Ag for 45 minutes in indicated number of experiments. (E) IgE-sensitized BMMCs were also evaluated for their ability to secrete CCL2 and IL6 after stimulation with IgE/Ag for 60 and 120 min as indicated. Data shown in (A–D) are the mean ± s.e.m of indicated experiments. Statistical analysis was done using an unpaired Student’s t test. Data in (E) have been normalized by setting WT values to 1. The range of maximal release was between 2,3 to 83,2 ng/mL for CCL-2 and 61,7 to 3781 pg/mL for IL-6. Statistical analysis was done using a Wilcoxon Test. No differences in basal secretion in the absence of Ag was noted between the genotypes: CCL2 WT 1,95 ± 0,98 ng/mL; IRAP KO 2,46 ± 0,63 ng/mL; p = 0,75 and IL6 WT 9,5 ± 6,6 pg/mL; IRAP KO 5,6 ± 4,0 pg/mL; p = 0,63; unpaired Student’s t test; *: P < 0.05; **: P < 0.01.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Flow Cytometry, Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: IRAP rapidly relocates to the plasma membrane and partly colocalizes with IgE after stimulation of mast cells with Ag. (A) WT BMMCs were sensitized with anti-DNP IgE and were then plated on fibronectin-coated glass coverslips. Cells were stimulated for indicated time points with antigen DNP-HSA. After fixation and permeabilization cells were stained with DAPI (blue), anti-IgE (green) and anti-IRAP (red) and analyzed by confocal microscopy. Images show representative sections with multiple cells for IgE and IRAP single staining as well as the merge of all colors. In the merge an insert with an enlarged cell is shown to evidence colocalization. (B) Quantitative analysis of plasma membrane recruitment was determined as described in Materials & Methods. (C) Quantitative analysis of colocalization of IRAP and IgE at the plasma membrane (PM) or (D) intracellularly following internalization of FcεRI-bound IgE. Note that internalization of FcεRI-bound IgE starts at 15 min. (B, C, D) Statistical analysis for plasma membrane recruitment and colocalization of IgE with IRAP was done using the one-way ANOVA followed by a Kruskal Willis post-test. Data shown are presented as the mean ± s.e.m of indicated individual cells (pooled from 3 experiments). *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Clinical Proteomics, Membrane, Staining, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: Diminished phosphorylation response of signaling effectors in FcεRI-stimulated BMMCs and IgG stimulated neutrophils and monocytes in the context of IRAP-deficiency. (A–C) WT and IRAP-deficient (IRAP KO ) BMMCs were sensitized with anti-DNP IgE for 24 hours before stimulating them with specific antigen (DNP-HSA). After indicated time points, stimulation was arrested by adding fixation and permeabilization buffer. Phosflow analysis of signaling effectors (left panel) was assessed using anti-pSyk Y519/520 (A) , anti-pLAT Y200 (B) and anti-pp38 Y182 (C) . Total levels of proteins in non-stimulated cells were also analyzed using anti-Syk, anti-LAT and anti p38 (right panels). Phosphorylation levels were determined as the ratio of the gMFI of samples divided by the gMFI of the FMO of the respective sample and represent the mean ± s.e.m of indicated experiments. Statistical analysis was done using a Student’s test. (D, E) Phosflow analysis of Syk phosphorylation at Y 519/520 was also determined on ex vivo IgG-stimulated neutrophils and monocytes during ASA. Cells were accessed directly from the blood 5 min after initiation of ASA. The gating strategies for analysis of BMMCs, neutrophils and monocytes and representative examples of Ab staining are shown, respectively, in ( Supplementary Figures 2B, C ). Data shown are analyzed as in (A–C) and presented as the mean ± s.e.m of indicated experiments. Statistical analysis was done using a Student’s test. *: P < 0.05; **: P < 0.01; *** P < 0.001. ns, not significant.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Phospho-proteomics, Ex Vivo, Staining

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: IRAP-deficient BMMCs show diminished phosphorylation of Syk at the plasma membrane where it colocalizes with IgE. (A) WT and IRAP-deficient (IRAP KO ) BMMCs were sensitized with anti-DNP IgE and challenged with antigen (30 ng/mL of DNP-HSA). Stimulation was arrested at indicated time points and cellular lysates were prepared in SDS sample buffer. Western Blot analysis (left panel) show pSyk staining (anti-pSyk Y519/520 ) and total Syk determined after stripping. Quantitative data (right panel) are the ratio between pSyk/Syk and were normalized by setting non stimulated values to 1. They represent the mean ± s.e.m of indicated experiments. Statistical analysis was done using the Wilcoxon test. (B) Anti-DNP IgE sensitized WT (blue) and IRAP KO BMMCs (red) were loaded with indo-1 and stimulated after addition (arrow) of 30 ng/mL of DNP-HSA. The fluorescence emission ratio for Ca 2+- bound/Ca 2+- free indo-1 was measured. Similar results were obtained in three independent experiments. (C) Anti-DNP IgE sensitized WT and IRAP KO BMMCs were plated on fibronectin-coated glass coverslips. Cells were stimulated with 30 ng/mL of DNP-HSA for indicated time points. After fixation and permeabilization cells were stained with DAPI (blue), anti-IgE (green) and anti-pSyk Y519/520 (red) as indicated. Cells were analyzed by confocal microscopy. Images show representative sections with multiple cells for IgE and pSyk Y519/520 single staining as well as the merge of all colors. In the merge an insert with an enlarged cell membrane is shown to evidence colocalization. For a complete kinetic analysis see ( Supplementary Figure 4 ) . (D) The total amount of pSyk Y519/520 in both genotypes was determined for all time points examined. (E) The amount of pSyk Y519/520 appearing at the plasma membrane within the first 15 min after stimulation was quantified. (F) The areas of colocalization between pSyk Y519/520 and IgE during the entire time of stimulation were quantified. (D–F) Data shown are presented as the mean ± s.e.m of indicated individual cells analyzed (pooled from 3 different experiments). Statistical analysis was done using the two-way ANOVA followed by Sidak’s post-hoc test. *: P < 0.05; **: P < 0.01; *** P < 0.001; **** P < 0.0001. ns, not significant.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Phospho-proteomics, Clinical Proteomics, Membrane, Western Blot, Staining, Stripping Membranes, Fluorescence, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: Absence of IRAP does not affect early signaling events mediated by Lyn kinase. (A) IgE-sensitized WT and IRAP-deficient (IRAP KO ) BMMCs were either not stimulated or stimulated with antigen (30 ng/mL of DNP-HSA) before being lysed and immunoprecipitated with anti-FcεRIβ chain mAb. Levels of co-immunoprecipitated phospho-β chain (**) and phospho-γ chain (*) in non-reduced gels were determined using anti-phospho-tyrosine (PY20) Ab. Total immunoprecipitated FcεRI receptors were determined after stripping and reblotting with anti-FcεRβ chain mAb. A representative blot and quantitative analysis of indicated number of experiments are shown in the left and right panels, respectively. Statistical analysis was done using a Student’s t-test. (B) WT and IRAP-deficient (IRAP KO ) BMMCs were sensitized with anti-DNP IgE and were then plated on fibronectin-coated glass coverslips. Cells were stimulated with 30 ng/mL of DNP-HSA for indicated time points. After fixation and permeabilization cells were stained with DAPI (blue), anti-IgE (green) and anti-Lyn (red) Abs as indicated. Cells were analyzed by confocal microscopy. Images show representative sections (out of 2 experiments) with multiple cells for IgE and Lyn single staining as well as the merge of all colors. ns, not significant.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Immunoprecipitation, Stripping Membranes, Staining, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation

doi: 10.3389/fimmu.2022.1029759

Figure Lengend Snippet: IRAP-deficient cells show less SHP1-inactivating phosphorylation on Ser591. (A) Anti-DNP IgE sensitized WT and IRAP-deficient (IRAP KO ) BMMCs were stimulated with specific antigen (30 ng/mL of DNP-HSA) for indicated time points and levels of the SHP1 S591 phosphorylation response were determined at indicated time points by phosflow analysis using anti-SHP1 S591 (left panel). Total levels of proteins of resting cells were analyzed using anti-SHP1 (right panel). (B, C) SHP1 phosphorylation on Ser591 was also determined on IgG-stimulated neutrophils (B) and monocytes (C) during ASA analyzed ex vivo using phosflow analysis. Cells were accessed directly from the blood 5 min after initiation of ASA. The gating strategies for analysis of BMMCs, neutrophils and monocytes and representative examples of Ab staining are shown, respectively in ( Supplementary Figures 2B, C ). Phosphorylation levels in (A–C) were determined as the ratio of the gMFI of samples divided by the gMFI of the Isotype of the respective sample and represent the mean ± s.e.m of indicated experiments. Statistical analysis was done using a Student’s test. *: P < 0.05; **: P < 0.01; ns, not significant.

Article Snippet: Abs used: IRAP (D7C5) XP ® Rabbit mAb (Cell signaling #6918S), LifeSpan Bioscience.

Techniques: Phospho-proteomics, Ex Vivo, Staining

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: Loss of BATF impairs Th17 differentiation. ( A ) Rpkm values are plotted for BATF RNA at different time points of activation (Th0) or Th17-differentiation, using our published RNA-seq data . ( B ) Immunoblot (left) shows BATF protein levels in SCR versus BATF KD cells, at 24 h of Th17 polarization. Actin serves as loading control. Adjoining flow cytometry plots show percentage of CCR6 positive cells and the graph below shows ELISA analysis for IL-17 secretion in SCR versus BATF KD cells, at 72 h of Th17 polarization. ELISA values were first normalized for cell count (live), and then normalized to SCR control. Graph shows mean ± SEM for three biological replicates. Statistical significance was calculated using two-tailed Student's t test (** p < 0.01). ( C ) Volcano plots highlight the significantly upregulated (in red) and downregulated (in blue) genes in BATF-silenced Th17 cells at 24 h (left) and 72 h (right) of polarization (FDR ≤ 0.1, |FC| ≥ 1.8). DE genes with relevance to Th17 function are shown (extended DE gene list shown in ). ( D ) IPA was used to identify pathways altered upon silencing of BATF in Th17-polarized cells (24 h and 72 h). The top pathways related to T-cells and immune signaling are selectively shown. ( E ) Venn diagram shows the overlap between the genes that are altered upon BATF KD and the genes whose putative promoter regions (5-kb window around the TSS) are bound by BATF. The overlapping area represents the promoter-bound regulatory targets of BATF and the adjoining heatmap shows their corresponding expression changes in BATF KD Th17 cells. IGV images illustrate the occupancy of BATF over some of its Th17-associated targets. ( F ) Figure shows the topmost consensus sequence for genomic-binding of BATF, and the top six TF motifs enriched within BATF-bound sites, which were obtained using de-novo motif enrichment analysis by Homer. Peaks with IDR p value <0.01 were used for motif discovery.

Article Snippet: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

Techniques: Activation Assay, RNA Sequencing, Western Blot, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Counting, Two Tailed Test, Expressing, Sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: Comparing transcriptional targets and genomic binding sites of FOSL proteins with BATF. ( A ) Heatmap on the top shows logarithmic FC values for the DE genes that show opposite expression changes in FOSL DKD and BATF KD Th17 cells, at the indicated time points of differentiation. Heatmap in the bottom panel depicts the DE genes that show similar expression changes in FOSL DOE and BATF KD Th17 cells. Th17-related genes are highlighted in red. ( B ) ChIP-seq profiles of FOSL1, FOSL2 and BATF in Th17 cells. Graph (above) shows the overlay between the peak distribution profiles of the three TFs. Bar plot (below) depicts peak-annotation results for their identified binding sites. ( C ) Heatmap with k-means clustering shows the ChIP-seq signal intensities ± 2-kb around the centers of the genomic-binding regions of FOSL1, FOSL2 and BATF. ( D ) Venn diagram shows an overlap between the genomic binding sites of FOSL1, FOSL2 and BATF (overlap represents peaks sharing 200 bp or more). Adjoining heatmap depicts Log2FC values for the gene targets that are co-bound and oppositely regulated by FOSL proteins and BATF, at the given time points of Th17 differentiation. Genes showing shared occupancy of the three factors over promoter regions have been marked (*asterisk). Th17-related targets are highlighted. ( E ) IGV track snapshots illustrate the co-localization of FOSL1, FOSL2 and BATF over selected Th17-linked genes. The profile of H3K27ac histone mark around the shared binding sites of the three factors is shown. ( F ) Bar plot depicts immunoblot-based expression analysis of STAT4 in FOSL DKD (left) and BATF KD (right) Th17 cells, cultured for 72 h. Data shows mean ± SEM for three or four biological replicates, as indicated. Statistical significance is calculated using two-tailed Student's t test (* p < 0.05). Adjoining IGV track shows the binding overlap of FOSL1, FOSL2 and BATF, flanked by H3K27ac marks near the STAT4 locus.

Article Snippet: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

Techniques: Binding Assay, Expressing, ChIP-sequencing, Western Blot, Cell Culture, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: BATF and FOSL proteins show common interacting partners in Th17 cells. ( A ) Figure illustrates the common binding partners of FOSL1 and FOSL2 in Th17 cells (72 h), based on data acquired from our recent study . Interactors having reported roles in T-cell function are shown. ( B ) STRING network analysis of human BATF. Width of lines between the nodes indicate confidence values for each protein-protein association. Interactions with a minimum score of 0.7 are shown (high confidence). ( C ) BATF was immunoprecipitated using 72 h-polarized Th17 cultures. Immunoblotting was then used to analyse its interaction with selected (shared) binding partners of FOSL1 and FOSL2 (JUNB, SIRT-1, JUN and RUNX1). Data is shown for three biological replicates. Immunoblot for BATF confirms immunoprecipitation of the factor.

Article Snippet: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

Techniques: Binding Assay, Cell Function Assay, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation

doi: 10.1093/nar/gkac256

Figure Lengend Snippet: SNPs associated with autoimmune diseases localize within the genomic binding sites of FOSL1, FOSL2 and BATF. ( A ) Enrichment of disease-associated SNPs (or their proxies in Caucasian populations) within FOSL1, FOSL2 and BATF genomic-binding sites, relative to random sets of background SNPs. ( B ) SNPs relevant to the study were shortlisted . Of these, the SNPs that were functionally validated in DNA-affinity precipitation assays are shown. ( C, D ) DAPA followed by immunoblot analysis shows the SNPs that alter the binding of FOSL1, FOSL2 or BATF to their genomic sites (identified by ChIP-seq analysis). Wildtype (WT) oligonucleotides containing the binding motifs of these TFs (at different genomic loci), and mutant oligonucleotides harbouring a SNP within the binding motif, were used as baits for pull-down of the corresponding AP-1 factor from 72 h Th17-polarized cell lysates. For experimental controls, an oligonucleotide with a conserved binding sequence for BATF (BATF WT), and the corresponding mutated sequence which is known to disrupt BATF occupancy (BATF MUT) were used. Panel C includes SNPs affecting the binding of either FOSL1, FOSL2 or BATF. Those SNPs at the common binding sites of the three factors which also alter the binding affinities for all of them are shown in panel D. The common SNPs harboured within consensus AP-1 motifs are labelled. Data is representative of three biological replicates.

Article Snippet: FOSL1, FOSL2 and BATF protein was analysed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4), rabbit monoclonal FOSL2 antibody (D2F1E) and rabbit monoclonal BATF antibody (D7C5) from Cell Signaling Technology.

Techniques: Binding Assay, Affinity Precipitation, Western Blot, ChIP-sequencing, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A and B ) Immunoblot shows BATF protein levels in control (SCR) versus BATF KD Th17 cells at 24h of differentiation. Adjoining flow cytometry plots depict the percentage of CCR6 positive cells in the indicated conditions, at 72h of Th17 polarization. Panel B shows ELISA analysis for secreted IL-17A levels in SCR versus BATF KD Th17 cells (72h). ELISA values were first normalized to live cell count, followed by normalization with SCR. Graph shows mean ± SEM for three biological replicates. Statistical significance was calculated using two-tailed Student’s t test (**p < 0.01). ( C ) Volcano plots highlight the significantly upregulated (red) and downregulated (blue) genes in BATF-silenced Th17 cells at 24h [left] and 72h [right] of polarization (FDR ≤ 0.1, |fold change| ≥ 1.8). DE genes with relevance to Th17 function are shown. ( D ) IPA was used to identify pathways altered upon silencing of BATF in Th17-polarized cells. ( E ) ChIP-seq analysis was performed for BATF using Th17 cells cultured for 72h. Figure shows distribution of BATF binding sites relative to the position of the closest transcription start site (TSS). TSS is defined to be at position zero. ( F ) The topmost consensus sequence for genomic-binding of BATF and the top six TF motifs enriched within BATF-bound sites, were obtained using de-novo motif enrichment analysis by Homer. Peaks with IDR p value < 0.01 were used for motif discovery. ( G ) Venn diagram shows the overlap between the genes that are altered upon BATF KD and the genes whose promoters are bound by BATF (5-kb around TSS). The overlapping area represents the promoter-bound regulatory targets of BATF and the adjoining heatmap shows their corresponding logarithmic fold change values in BATF KD Th17 cells. IGV images illustrate the occupancy of BATF over some of its Th17-associated targets.

Article Snippet: FOSL1, FOSL2 and BATF protein was analyzed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4; 1:500), rabbit monoclonal FOSL2 antibody (D2F1E; 1:1000) and rabbit monoclonal BATF antibody (D7C5; 1:1000) from Cell Signaling Technology.

Techniques: Western Blot, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Counting, Two Tailed Test, ChIP-sequencing, Cell Culture, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Heatmaps show top DE genes (FDR ≤ 0.1, |fold change| ≥ 1.8) in BATF-silenced Th17 cells at 24h [left] and 72h [right] of polarization. Scaled expression values are plotted and genes associated with Th17 cell-function are highlighted (upregulated genes are in red, downregulated genes in blue). ( B and C ) Panel B shows flow cytometry analysis for percentage of CCR6 positive cells in non-targeting versus BATF-silenced Th17 cells, at 72h of polarization. Panel C depicts qRT-PCR analysis for IL-17A [left] and IL-17F [right] transcript levels under the mentioned conditions. Data shows mean ± SEM for three biological replicates. Statistical significance was calculated using two-tailed Student’s t test (*p < 0.05; **p < 0.01). ( D ) Western blot analysis shows STAT4, RORC and BATF protein levels in non-targeting versus BATF KD Th17 cells, at 72h of polarization. Data for three biological replicates is shown and the quantified bar plot is provided as a part of .

Article Snippet: FOSL1, FOSL2 and BATF protein was analyzed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4; 1:500), rabbit monoclonal FOSL2 antibody (D2F1E; 1:1000) and rabbit monoclonal BATF antibody (D7C5; 1:1000) from Cell Signaling Technology.

Techniques: Expressing, Cell Function Assay, Flow Cytometry, Quantitative RT-PCR, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Heatmap on the top shows logarithmic fold-change values for the DE genes that are oppositely regulated in FOSL DKD and BATF KD Th17 cells, at the indicated time points of polarization. Heatmap in the bottom panel depicts the DE genes that are similarly altered in FOSL DOE and BATF KD Th17 cells. Th17-relevant genes are highlighted in red. ( B ) Comparing the ChIP-seq profiles of FOSL1, FOSL2 and BATF in Th17 cells. Graph (above) shows the overlay between the peak distribution profiles of the three TFs. Bar plot (below) depicts peak-annotation results for their identified binding sites. ( C ) Heatmap with k-means clustering shows the ChIP-seq signal intensities ± 2-kb around the centers of the genomic-binding regions of FOSL1, FOSL2 and BATF. Th17-associated genes in the vicinity of the binding sites are highlighted within the respective clusters. ( D ) Venn diagram shows an overlap between the genomic binding sites of FOSL1, FOSL2 and BATF (overlap represents peaks sharing 200 bp or more). Adjoining heatmap depicts fold-change values for the gene targets that are co-bound and oppositely regulated by FOSL proteins and BATF. Genes showing shared occupancy of the three factors over putative-promoters have been marked (*asterisk). Th17-relevant targets are highlighted. ( E ) IGV track snapshots illustrate the co-localization of FOSL1, FOSL2 and BATF over selected Th17 genes. Profile of H3K27ac marks around the shared sites is shown. ( F ) Bar plot depicts immunoblot-based expression analysis of STAT4 in FOSL DKD [left] and BATF KD [right] Th17 cells, cultured for 72h. Data shows mean ± SEM for three or four biological replicates, as indicated. Statistical significance is calculated using two-tailed Student’s t test (*p < 0.05). Adjoining IGV track shows the binding overlap of FOSL1, FOSL2 and BATF, flanked by H3K27ac marks near the STAT4 locus.

Article Snippet: FOSL1, FOSL2 and BATF protein was analyzed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4; 1:500), rabbit monoclonal FOSL2 antibody (D2F1E; 1:1000) and rabbit monoclonal BATF antibody (D7C5; 1:1000) from Cell Signaling Technology.

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Expressing, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Figure illustrates the common binding partners of FOSL1 and FOSL2 in Th17 cells (72h), based on data acquired from a parallel study of our lab ( bioRxiv ). Interactors having reported roles in T-cell function are shown. ( B ) STRING network analysis of human BATF. Width of lines between the nodes indicate confidence values for each protein-protein association. Only interactions with a minimum score of 0.7 are shown (high confidence). ( C and D ) Immunoprecipitated BATF was analyzed for its interaction with selected common binding partners of FOSL1 and FOSL2 (JUNB, SIRT-1, JUN and RUNX1), using western blotting (panel C). Additionally, BATF-interaction with STAT3 and IRF4 was analyzed to validate their previously-known association in mouse (panel D). Data is shown for three biological replicates.

Article Snippet: FOSL1, FOSL2 and BATF protein was analyzed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4; 1:500), rabbit monoclonal FOSL2 antibody (D2F1E; 1:1000) and rabbit monoclonal BATF antibody (D7C5; 1:1000) from Cell Signaling Technology.

Techniques: Binding Assay, Cell Function Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Enrichment of disease-associated SNPs (or their proxies in Caucasian populations) within FOSL1, FOSL2 and BATF genomic-binding sites, relative to random sets of background SNPs. ( B ) SNPs relevant to the study were shortlisted. Out of these, the SNPs that were functionally validated in DNA-affinity precipitation assays (DAPA) have been shown. ( C and D ) DAPA reveals the SNPs that alter the binding of FOSL1, FOSL2 or BATF to their genomic sites that were identified by ChIP-seq analysis. Wildtype (WT) oligonucleotides containing the binding motifs of these TFs (at different genomic loci), and mutant oligonucleotides harboring a SNP within the corresponding motif, were used as baits. For experimental controls, an oligonucleotide with a conserved binding sequence for BATF (BATF WT), and the corresponding mutated sequence which is known to disrupt BATF occupancy (BATF MUT) were used. Immunoblot results for the SNPs unique to FOSL1, FOSL2 and BATF (panel C), and the ones common across the three factors (panel D) are shown. Data is representative of three biological replicates.

Article Snippet: FOSL1, FOSL2 and BATF protein was analyzed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4; 1:500), rabbit monoclonal FOSL2 antibody (D2F1E; 1:1000) and rabbit monoclonal BATF antibody (D7C5; 1:1000) from Cell Signaling Technology.

Techniques: Binding Assay, Affinity Precipitation, ChIP-sequencing, Mutagenesis, Sequencing, Western Blot

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Table illustrates information on the autoimmune-linked SNPs that are harbored within consensus AP-1 motifs at the shared genomic-binding sites of FOSL1, FOSL2 and BATF. The sequence logos shown have been derived from the respective TF ChIP-seq peaks using Homer. ( B and C ) DAPA analysis was performed to test the effect of selected SNPs on the DNA-binding abilities of FOSL1, FOSL2 and BATF. The immunoblot images in panels B & C show biological replicates (R1, R2, R3) for and respectively

Article Snippet: FOSL1, FOSL2 and BATF protein was analyzed by western blotting using rabbit monoclonal FOSL1 antibody (D80B4; 1:500), rabbit monoclonal FOSL2 antibody (D2F1E; 1:1000) and rabbit monoclonal BATF antibody (D7C5; 1:1000) from Cell Signaling Technology.

Techniques: Binding Assay, Sequencing, Derivative Assay, ChIP-sequencing, Western Blot