Journal: Infection and Immunity

Article Title: Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii

doi: 10.1128/IAI.00376-19

Figure Lengend Snippet: SSChigh granulocytes express CD11b and the interleukin-5 receptor. BALB/c mice were vaccinated s.c. with 10 μg of PIV or PIIV, and spleens were processed for flow cytometry as previously described. Mice receiving Alhydrogel adjuvant alone served as unvaccinated controls. Naïve mice were used to establish baseline values. (A) Gating strategy. FSC and SSC were used to remove cell debris from the analysis. CD45+ leukocytes were then identified and further subgated based on SSC and expression of CD11b. CD11b+ SSChigh granulocytes were then further subgated based on expression of the interleukin-5 receptor alpha subunit (IL-5Rα, CD125). (B and C) Population frequencies and absolute cell counts were determined at 7 and 14 dpv. Each experimental group includes 4 to 6 mice, with error bars representing the standard deviation from the mean. *, P < 0.05; **, P < 0.01, as determined by one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Recombinant anti-mouse CD125 (IL-5Ra, clone REA343; conjugated to phycoerythrin [PE]), Siglec-F (clone REA798; PE-Vio770), and CD45R (B220, clone REA755; fluorescein isothiocyanate [FITC]) as well as rat anti-mouse CD19 (clone 6D5; FITC) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Adjuvant, Expressing, Standard Deviation, Comparison

Journal: Infection and Immunity

Article Title: Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii

doi: 10.1128/IAI.00376-19

Figure Lengend Snippet: SSChigh CD11b+ CD125+ cells are Siglec-F+ eosinophils. BALB/c mice were vaccinated s.c. with 10 μg of PIV or PIIV, and spleens were processed for flow cytometry. Mice receiving Alhydrogel adjuvant alone served as unvaccinated controls. Naïve mice were used to establish baseline values. (A) Gating strategy. SSChigh CD11b+ CD125+ cells were examined for expression of Siglec-F (eosinophils) and CD19/B220 (B cells). (B and C) Population frequencies and absolute cell counts were determined at 7 and 14 dpv. Each experimental group includes 4 to 6 mice, with error bars representing the standard deviations from the means. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Recombinant anti-mouse CD125 (IL-5Ra, clone REA343; conjugated to phycoerythrin [PE]), Siglec-F (clone REA798; PE-Vio770), and CD45R (B220, clone REA755; fluorescein isothiocyanate [FITC]) as well as rat anti-mouse CD19 (clone 6D5; FITC) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Adjuvant, Expressing, Comparison

Journal: Infection and Immunity

Article Title: Eosinophils Affect Antibody Isotype Switching and May Partially Contribute to Early Vaccine-Induced Immunity against Coxiella burnetii

doi: 10.1128/IAI.00376-19

Figure Lengend Snippet: Eosinophil accumulation in PIV-vaccinated mice is CD4+ T cell dependent. WT C57BL/6 and CD4-deficient mice were vaccinated s.c. with 10 μg of PIV, and 7 days later spleens were harvested for flow cytometry or ex vivo restimulation studies. Mice receiving Alhydrogel adjuvant alone served as unvaccinated controls. Naïve mice were used to establish baseline values. (A) SSChigh CD11b+ CD125+ Siglec-F+ eosinophils were enumerated using the gating strategy described in the legends for Fig. 4 and ​and5.5. (B) Total splenocytes were isolated and restimulated with medium or with 1 × 107 C. burnetii NMI bacteria for 3 and 6 days, and cell culture supernatants were evaluated for IFN-γ production by cytokine-specific ELISA. Each experimental group includes 5 mice, with error bars representing the standard deviations from the means. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, as determined by one-way ANOVA with Tukey’s multiple-comparison test. Cb, C. burnetii; KO, knockout.

Article Snippet: Recombinant anti-mouse CD125 (IL-5Ra, clone REA343; conjugated to phycoerythrin [PE]), Siglec-F (clone REA798; PE-Vio770), and CD45R (B220, clone REA755; fluorescein isothiocyanate [FITC]) as well as rat anti-mouse CD19 (clone 6D5; FITC) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Ex Vivo, Adjuvant, Isolation, Bacteria, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison, Knock-Out

Journal: Frontiers in Immunology

Article Title: Local Innate Markers and Vaginal Microbiota Composition Are Influenced by Hormonal Cycle Phases

doi: 10.3389/fimmu.2022.841723

Figure Lengend Snippet: Antibodies used to characterize the neutrophil subpopulations.

Article Snippet: CDw125 , REA705 , PE , 2.5 μL , 130-110-544 , Milteny.

Techniques: Clone Assay, Concentration Assay

Journal: Respiratory Research

Article Title: IL-5 signaling in asthmatic derived fibroblasts exacerbates airway remodeling through ECM dysregulation and apoptosis resistance

doi: 10.1186/s12931-025-03371-x

Figure Lengend Snippet: IL-5 inducing the expression of IL-5Ra in lung derived fibroblasts. A mRNA levels of IL5RA in both normal and asthmatic derived lung fibroblasts. 18s was used as a housekeeping gene for ΔCt normalization, and fold changes upon IL-5 treatment compared to control was calculated as 2. −ΔΔct . Data shown are the mean ± SD of four separate experiment; two-tailed paired student’s t-Test statistical analysis was done * p < 0.05, ** p < 0.01, *** p < 0.001 . B Cultured normal and asthmatic fibroblasts, captured at 40X magnification, display immunofluorescence staining for IL-5Rα (green), vimentin (red), and DAPI (blue) in both cell types, along with mouse and rabbit IgG isotype controls. C Evaluation of the mean fluorescence intensity (MFI) of IL-5Rα expression in lung fibroblasts, quantified following 24 h of IL-5 stimulation. Statistical significance was determined using two-tailed paired Student's t-test based on data from three independent experiments, * p < 0.05, ** p < 0.01, and *** p < 0.001

Article Snippet: Subsequently, the cells were incubated overnight at 4 °C with primary antibodies against IL-5Rα (Cat: MAB253, R&D Systems) Vimentin (Cat: 5741, Cell Signalling Technology), Rabbit mAb IgG XP® Isotype Control (Cat: 3900, CST), Mouse IgG isotype control (Cat: MAB002, R&D systems) at a 1:100 dilution.

Techniques: Expressing, Derivative Assay, Control, Two Tailed Test, Cell Culture, Immunofluorescence, Staining, Fluorescence

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 2. Heterodimerization of the Cytoplasmic Parts of LEPRb and LEPRa Does Not Induce STAT3 Activation A, COS-7 cells were transfected with expression plasmids encoding the IL-5R and chimeras of LEPRb, LEPRa, gp130, and gp130 without cytoplasmic part (gp130cyt), as indicated. Untransfected cells were supplemented with transfection reagent alone. Cells were cotransfected with 0.3 g of STAT3 expression vector per 6-cm dish. Two days after transfection, cells were stimulated with IL-5 (80 ng/ml) for 30 min before nuclear extracts were prepared. Nuclear extracts were analyzed by Western

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 3. JAK2 Association with gp130, LEPRa, and LEPRb COS-7 cells were cotransfected with expression con- structs encoding the indicated receptor chimeras (/gp130, /LEPRa, /LEPRb, or /gp130box1) and JAK2. After mild lysis of the cells, receptor complexes were immunoprecipi- tated with the anti-IL-5R antibody S-16. Immunocomplexes or cellular lysates were immunoblotted with antibodies against JAK2 or IL-5R as indicated. Observations were con- firmed in four independent experiments.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Expressing, Lysis

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 4. Structure of the Chimeric Receptor Constructs Used in This Study A, Intracellular amino acid sequences of CLRa and CLRb. Presumed box 1 and box 2 motifs (17, 35, 36) are underlined (closed lines); the unique amino acids in the C terminus of CLRa are marked by a dashed line. IL-5R extracellular parts are illustrated as gray boxes; extracellular and transmembrane (TM) regions of the leptin receptor are indicated as white boxes. The positions where stop codons were inserted into CLRb to generate the indicated deletion mutants are illustrated by vertical lines. K889 of LEPR is the last amino acid of CLR-0. B, Schematic representation of deletion mutants of CLRb (in comparison to CLRa and CLRb). The splice site (after amino acid K889) is illustrated by a dashed line. The box 1–3 motifs are represented by black boxes. The membrane proximal 29 amino acids of the cytoplasmic part of the LEPR are identical in LEPRa and LEPRb and include a box 1 motif. Each construct is designated by the number of unique C-terminal amino acids. The amino acids unique in CLRa are illustrated by a hatched box. The deletion constructs contain no intracellular tyrosine residue. Lengths of receptor constructs are not drawn to scale.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Construct, Comparison, Membrane, Residue

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 5. Signaling of Deletion Mutants of CLRb in Heterodimeric Complexes with CLRb A and B, COS-7 cells were cotransfected with expression plasmids encoding the indicated chimeric receptor constructs and a STAT3 expression vector. Thirty-two hours after transfection, cells were starved for 16 h and then treated with 80 ng/ml IL-5 or vehicle only for 30 min before nuclear extracts were prepared. Extracts were used for Western blot analysis with a phospho-specific STAT3 antibody. For expression control, lysates were probed with antibodies against STAT3, human (h)IL-5R, and hIL-5R. C, HepG2 cells were transfected with expression plasmids encoding /CLRb and the indicated chimera along with an 2-macroglobulin promoter luciferase reporter gene construct. One day after transfection, cells were stimulated with IL-5 (80 ng/ml) or vehicle only for 24 h. Luciferase activity of lysates was normalized to the activity of coexpressed -galacto- sidase. One representative experiment is shown. Columns represent the average of two independent transfections and bars reflect the differences between the two. The same differences between the average values of the expression constructs were observed in three additional experiments.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Expressing, Construct, Plasmid Preparation, Transfection, Western Blot, Control, Luciferase, Activity Assay

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Identification of the critical sequence elements in the cytoplasmic domain of leptin receptor isoforms required for Janus kinase/signal transducer and activator of transcription activation by receptor heterodimers.

doi: 10.1210/mend.16.4.0800

Figure Lengend Snippet: Fig. 7. Mutational Analysis of a 13-Amino Acid Region Crucial for STAT3 Signaling by CLRb A, Design of point mutants from CLR-15 and CLR-19. Sequences of the membrane proximal cytoplasmic segments of the LEPR of Mus musculus (EMBL database accession no. Y10298; upper sequence) and Gallus gallus (AF169827; lower sequence) are aligned. In CLR-15 and CLR-19, conserved amino acids were replaced by alanine (arrows). The box 1 motif (solid lines) and the proposed box 2 motif (dashed lines) (17) are boxed. The transmembrane region (TM) is indicated. B, COS-7 cells were transfected with expression plasmids encoding the IL-5R and chimeras as indicated and a STAT3 expression vector. The experiment was performed as described in Fig. 5. Results for each construct were confirmed in three independent experiments.

Article Snippet: The following primary antibodies were used: anti-phospho-STAT3 (Tyr705) and anti-STAT3 rabbit polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), antimouse LEPR goat antibody from R&D Systems (Wiesbaden, Germany), antihuman IL-5R (N-20) rabbit polyclonal antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antihuman IL-5R goat antibody from R&D Systems, anti-pTyr antibodies PY99 from Santa Cruz Biotechnology, Inc., 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and PY20 from Transduction Laboratories, Inc. (Lexington, KY).

Techniques: Membrane, Sequencing, Transfection, Expressing, Plasmid Preparation, Construct