Journal: Immunity

Article Title: Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

doi: 10.1016/j.immuni.2020.07.006

Figure Lengend Snippet:

Article Snippet: anti-pSTAT6 149 Sm-conjugated Clone 18 , Fluidigm , Cat No.3149004A.

Techniques: Recombinant, Virus, Molecular Cloning, Blocking Assay, Conjugation Assay, Staining, Western Blot, Lysis, Extraction, Mutagenesis, Isolation, Reverse Transcription, Luminex, Transfection, Amplification, Software, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4

doi: 10.3390/ijms25031931

Figure Lengend Snippet: The STAT6 phosphorylation HT29 cell-based assay indicated that the acidic species had 3.6-fold higher potency than the main species. HT29 pSTAT6 assay was performed as described in .

Article Snippet: Fixed cells were stained with PE-labeled antibody to human pSTAT6 (pY641, Clone 18; BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Cell Based Assay

Journal: International Journal of Molecular Sciences

Article Title: Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4

doi: 10.3390/ijms25031931

Figure Lengend Snippet: Summary of IC50 values for the anti-IL-4 antibody 3B9 species in neutralization of IL-4 induced pSTAT6 formation in HT-29 cells.

Article Snippet: Fixed cells were stained with PE-labeled antibody to human pSTAT6 (pY641, Clone 18; BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Neutralization

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Representative image demonstrates the peri-infarct area defined by Iba1 staining. Two randomly selected microscopic fields in the cortex (CTX) and 2 in the striatum (STR) of each section were subjected for analysis. Scale bar: 200 μm. (B) Representative images of coronal brain sections showing the phosphorylation of STAT6 (green) in Iba1+ microglia/macrophages (red) at indicated time points after tMCAO. Scale bar: 20 μm. (C) Representative images of coronal brain slices collected 3d after tMCAO showing the nuclear (DAPI, blue) localization of pSTAT6 (green) in Iba1+ (red) microglia/macrophages. Scale bar: 20 μm. (D) Quantification of the number of pSTAT6+Iba1+ cells in the ischemic regions indicated in B at different time points after tMCAO. n = 3–4 mice per group. **P ≤ 0.01, ***P ≤ 0.001 vs. 1d in CTX. ##P ≤ 0.01 vs. 1d in STR, 1-way ANOVA. (E and F) Flow cytometric analysis of pSTAT6 in brain cells 3d after tMCAO. (E) Representative dot plots demonstrate the gating strategy for microglia/macrophages (CD45+CD11b+), astrocytes (CD45–GLAST+), oligodendrocytes (CD45–O4+), and neurons (CD45–NeuN+). (F) Mean fluorescence intensity (MFI) of pSTAT6 in brain cells. n = 3 mice. ***P ≤ 0.01, Student’s t test. (G) Representative plot of STAT6 activation (pSTAT6) in microglia (CD45intermediate; CD45int) and macrophages (CD45hi) in pSTAT6+CD11b+ population. (H) STAT6 activation evaluation in microglia and macrophages by MFI of pSTAT6. n = 3 mice. **P ≤ 0.01, Student’s t test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Staining, Fluorescence, Activation Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Brains were collected from WT and STAT6-KO mice 3d and 7d after 60 minutes tMCAO. (A–D) Analysis of microglia/macrophage phenotypes. (A) Representative images of microglia/macrophage marker Iba1 (red) and proinflammatory phenotype marker CD16 (green) double-staining. Scale bar: 50 μm. (B) Representative images of microglia/macrophage marker Iba1 (red) and antiinflammatory phenotype marker CD206 (green) double-staining. Scale bar: 50 μm. (C) Quantification of Iba1+CD16+ proinflammatory microglia/macrophages in ischemic areas at 3d and 7d after tMCAO. (D) Quantification of Iba1+CD206+ antiinflammatory microglia/macrophages in ischemic areas at 3d and 7d after tMCAO. n = 3–6 mice per group. **P ≤ 0.01, ***P ≤ 0.001 STAT6-KO vs. WT, Student’s t test. (E) The expression of CD16 and CD206 in pSTAT6+ and pSTAT6– microglia/macrophages (CD45+CD11b+) in ipsilateral hemisphere from WT mice was measured by flow cytometry 3d after tMCAO. Mean fluorescent intensity (MFI) of CD16 or CD206 in pSTAT6–CD45+CD11b+ (gray) and pSTAT6+CD45+CD11b+ (pink) populations was quantified. n = 5 per group. ***P ≤ 0.001 pSTAT6+ vs. pSTAT6– population, Student’s t test. (F) mRNA expression of proinflammatory and antiinflammatory markers were measured by RT-qPCR in the ipsilateral hemisphere 3d and 7d after ischemic stroke. n = 6 mice per group for 3d. n = 3 mice per group for 7d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 STAT6-KO vs. WT, Student’s t test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Marker, Double Staining, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Schematic illustration for the construction of WT/CX3CR1-GFP chimeric mice. (B–D) ImageStream analysis of pSTAT6 and Arg1 in CD45+CD11b+GFP– cells and CD45+CD11b+GFP+ macrophages in ipsilateral hemisphere of chimeric mice was performed 3d after tMCAO. (B) Representative images indicating pSTAT6 and Arg1 signal in both CD45+CD11b+GFP– cells and CD45+CD11b+GFP+ macrophages in ipsilateral brains. Scale bar: 7 μm. (C) Percentages of GFP– cells and GFP+ macrophages among CD45+CD11b+ cells were assessed in WT (left) and WT/CX3CR1-GFP chimeric mice (right). (D) Percentages of pSTAT6+ cells (left) and Arg1+ cells (right) observed among CD45+CD11b+GFP– cells and CD45+CD11b+GFP+ macrophages. n = 3 mice. (E) Chimeric mice were constructed by transplanting WT BM to WT recipients (WT/WT), STAT6-KO BM to WT recipients (WT/KO), or WT BM to STAT6-KO recipients (KO/WT) after irradiation of recipients. Chimeric mice were subjected to 60 minutes of tMCAO at 6 weeks after irradiation. (F and G) Infarct volume at 3d after tMCAO was quantified in MAP2-stained (green) coronal sections. Dashed lines outline the infarct area. n = 9–10 mice per group. Scale bar: 1 mm. (H and I) Neuronal death in chimeric mice was quantified by TUNEL (red) and NeuN (green) colabeling in infarct areas 3d after tMCAO. Scale bar: 10 μm. n = 3–5 mice per group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. WT/WT, 1-way ANOVA.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Construct, Irradiation, Staining, TUNEL Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Immunostaining of Arginase 1 (Arg1, green) in Iba1+ microglia/macrophages (red) in the ischemic striatum (STR) and cortex (CTX) 3d after cerebral ischemia. Scale bar: 20 μm. (B) The expression of Arg1 in pSTAT6+ (pink) and pSTAT6– (gray) microglia/macrophage (CD45+CD11b+ gated) was measured 3d after tMCAO in ipsilateral hemisphere of WT mice by flow cytometry. Mean fluorescent intensity (MFI) of Arg1 was quantified. n = 5 mice. ***P ≤ 0.001 vs. pSTAT6– population, Student’s t test. (C) RT-qPCR analysis of Arg1 mRNA expression in WT and STAT6-KO microglia or macrophages without stimulation and after activating STAT6 signaling with IL-4 (20 ng/mL for 48 hours). Data were collected from 4 independent experiments. **P ≤ 0.01, *** P ≤ 0.001, 1-way ANOVA. (D) Western blot analysis of Arg1 protein expression in microglia or macrophages without stimulation and after activating STAT6 signaling with IL-4. Data were collected from 3 independent experiments. ***P ≤ 0.001, 1-way ANOVA.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Immunostaining, Expressing, Flow Cytometry, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma

doi: 10.1101/2022.11.28.518173

Figure Lengend Snippet: A . Examples of HFF cells stained with anti-pSTAT6 and Hoechst (to visualize DNA) after treatment with mycalolide B-treated parasites. B–C . Anhydrotetracycline (ATc) treatment did not significantly change the ability of MIC2-cKD or AMA1 -cKD parasites to elicit pSTAT6-positive host cell nuclei (B). However, ATc treatment significantly reduced the ability of MIC8-cKD parasites to elicit pSTAT6-positive host cell nuclei (C). Mean is plotted for n = 3 biological replicates; p values derive from one-way ANOVA followed by Sidak’s multiple comparison test prior to data normalization. D–F . All members of the CLAMP complex are required for robust nuclear translocation of host pSTAT6. Mean is plotted for n = 3 biological replicates; p values derive from one-way ANOVAs followed by Sidak’s multiple comparison tests prior to data normalization. G . The P. falciparum conditional knockdown system used to downregulate CLAMP or SPATR. Removal of ATc from the culture medium allows transcript binding of the TetR-DOZI fusion protein to the Tet aptamer sequences and leads to conditional loss of protein translation. H–K . Conditional knockdown of Pf CLAMP (H–I) or Pf SPATR (J–K) by removal of ATc from the culture medium leads to complete loss of parasite growth. Activity of constitutively expressed Renilla luciferase was used to track growth of each P. falciparum conditional knockdown line. Dihydroartemisinin (DHA) is used as a control for growth inhibition (I and K); p values derive from multiple t -tests for each time point using the Benjamini-Hochberg FDR approach (H and J) or one-way ANOVA followed by Sidak’s multiple comparison test (I and K).

Article Snippet: Cells were stained using anti-pSTAT6 (Cell Signalling cat. no. 9361S or Abcam cat. no. ab188080), anti-CDPK1 ( Waldman et al , 2020 ), and Hoechst (Santa Cruz cat. no. 394039).

Techniques: Staining, Translocation Assay, Binding Assay, Activity Assay, Luciferase, Inhibition

Journal: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-human phospho-STAT6 (Tyr641) (clone 18) , Fluidigm , Cat#3168012A; RRID:AB_2811103.

Techniques: Recombinant, Mass Cytometry, Software