pstat3-pe antibody ebioscience #501122408  (Thermo Fisher)

Journal: bioRxiv

Article Title: An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma

doi: 10.1101/2023.03.03.530773

Figure Lengend Snippet: A) Transcription of STAT3, SOCS3 and BCL3 in cells in cluster MatNeu2 comparing control individuals and NDMM patients B) Transcription of genes associated with STAT3-signaling in neutrophils cultured on MSC or on iMSC (n = 3 donors, collected in single experiment). HD, healthy donor C) Representative histogram of expression of phosphorylated STAT3 (pSTAT3) from CD10 + neutrophils cultured in the presence of G-CSF (positive control), cultured alone, cultured on non-inflammatory stroma (‘MSC’), or cultured on iMSC, as determined by flow cytometry. Fold change in mean fluorescent intensity (MFI) compared to neutrophils cultured alone (dotted line) was quantified per individual of each condition. Lines depict paired samples (n = 6, collected over 6 experiments) D) Frequencies of CD10 + neutrophils expressing C3AR, CD11b(act), and CD11c, or mean fluorescent intensity (MFI) of CD66b and CD45 on CD10 + neutrophils cultured on non-inflammatory stroma (‘+ MSC’) or on iMSC in the presence of DMSO or Stattic. Lines depict paired samples (n = 6, collected over 3 experiments) E) BAFF protein in supernatant of neutrophils cultured on non-inflammatory stroma (‘+ MSC’) or on iMSC in the presence of DMSO or Stattic. Lines depict paired samples (n = 6, collected over 3 experiments). F) IL-1β protein in supernatant of neutrophils cultured on non-inflammatory stroma (‘+ MSC’) or on iMSC in the presence of DMSO or Stattic. Lines depict paired samples (n = 5, collected over 3 experiments) G) Volcano plot depicting differentially expressed genes of MSC cultured with iMSC-conditioned neutrophils versus MSC cultured with MSC-conditioned neutrophils. Log 2 FoldChange cutoff, 1; adjusted p-value (padj) cutoff 10 -3 . Genes in red are related to iMSCs. (n = 5, collected over 3 experiments) H) Transcription (transcripts per million, TPM) of iMSC-related genes in MSC cultured with MSC-conditioned neutrophils (green) and MSC cultured with iMSC-conditioned neutrophils (blue). Dotted lined depict paired samples. (n = 5, collected over 3 experiments) I) Enrichment plots for the iMSC-signature (generated from DEGs of single cell RNA sequencing results in 4 ), and iMSC-like cells signature (generated from data in ) comparing MSC cultured with iMSC-conditioned neutrophils and MSC-conditioned neutrophils J) Transcription of iMSC-related genes in MSC cultured with iMSC-conditioned neutrophils in the presence of anti-IL-1β (grey) and in the presence of an isotype control (orange). Dotted lined depict paired samples (n = 5, collected over 3 experiments) K) Enrichment plots for the iMSC-signature (generated from DEGs of single cell RNA sequencing results in 4 ), and iMSC-like cells signature (generated from data in ) comparing MSC cultured with iMSC-conditioned neutrophils in the presence of anti-IL-1β or in the presence of an isotype control. Data are presented as mean ± SEM. Significance was calculated in B , G , H and J using the Wald test (two-tailed) followed by a Benjamini– Hochberg correction, and in C , D , E and F using the Wilcoxon Rank Sum test (two-tailed); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, NS P > 0.05.

Article Snippet: After permeabilisation, cells were washed twice and stained with pSTAT3[Tyr705]-PE (1:10; 13A3-1, BioLegend) at room temperature.

Techniques: Control, Cell Culture, Expressing, Positive Control, Flow Cytometry, Generated, RNA Sequencing, Two Tailed Test

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Activation of phosphorylation of STAT3 in HepG2 cells by hIL-6 or its mutants. HepG2 cells were treated with 50 ng/mL hIL-6 or its mutants for 15 min, and cell lysates were analyzed to detect phosphorylation of STAT3. Untreated cells were used as the negative control (NC). ( A ) Representative, digital western blot images of expression of P-STAT3, STAT3 and β-Actin upon stimulation with hIL-6 WT or R167A, E171A and R178E mutants, respectively; ( B ) statistical analysis of hIL-6 mutants compared with hIL-6 WT in term of STAT3 phosphorylation levels in HepG2 cells, based on three independent experiments. Data is presented as mean ± SD, n = 3. **p < 0.01; and ***p < 0.001.

Article Snippet: The antibody, PE labeled anti-pSTAT3 (pY705) monoclonal antibody was purchased from BD (Cat. 612569), and horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody was obtained from Southern Biotech (Cat. 2049–5).

Techniques: Activation Assay, Negative Control, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Activate of phosphorylation of STAT3 in Leukocytes by IL-6 mutants. Human leukocytes were preincubated with 50 ng/mL IL-6 mutants for 15 min, followed by permeabilized and stained. Untreated cells were used as the negative control (NC). Phosphorylation of STAT3 was assessed using flow cytometry with PE labeled anti-pSTAT3 (pY705) monoclonal antibody. ( A – C ) Gating strategy and representative histogram graph of P-STAT3 + cells.; ( D ) statistical analysis of percentage of P-STAT3 + cells upon stimulation with hIL-6 WT or R167A, E171A and R178E mutants, based on three independent experiments. The data is presented as mean ± SD, n = 3. *p < 0.05; **p < 0.01.

Article Snippet: The antibody, PE labeled anti-pSTAT3 (pY705) monoclonal antibody was purchased from BD (Cat. 612569), and horseradish peroxidase (HRP)-conjugated goat anti-human IgG antibody was obtained from Southern Biotech (Cat. 2049–5).

Techniques: Staining, Negative Control, Flow Cytometry, Labeling

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Activation of phosphorylation of STAT3 in HepG2 cells by hIL-6 or its mutants. HepG2 cells were treated with 50 ng/mL hIL-6 or its mutants for 15 min, and cell lysates were analyzed to detect phosphorylation of STAT3. Untreated cells were used as the negative control (NC). ( A ) Representative, digital western blot images of expression of P-STAT3, STAT3 and β-Actin upon stimulation with hIL-6 WT or R167A, E171A and R178E mutants, respectively; ( B ) statistical analysis of hIL-6 mutants compared with hIL-6 WT in term of STAT3 phosphorylation levels in HepG2 cells, based on three independent experiments. Data is presented as mean ± SD, n = 3. **p < 0.01; and ***p < 0.001.

Article Snippet: Subsequently, the cells were washed and stained for 30 min at room temperature in the dark with anti-pSTAT3 (pY705)-PE and an anti-mouse immunoglobulin G isotype-matched control (BD, Cat, 558595).

Techniques: Activation Assay, Negative Control, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Unveiling novel insights into human IL-6 − IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis

doi: 10.1038/s41598-024-69429-w

Figure Lengend Snippet: Activate of phosphorylation of STAT3 in Leukocytes by IL-6 mutants. Human leukocytes were preincubated with 50 ng/mL IL-6 mutants for 15 min, followed by permeabilized and stained. Untreated cells were used as the negative control (NC). Phosphorylation of STAT3 was assessed using flow cytometry with PE labeled anti-pSTAT3 (pY705) monoclonal antibody. ( A – C ) Gating strategy and representative histogram graph of P-STAT3 + cells.; ( D ) statistical analysis of percentage of P-STAT3 + cells upon stimulation with hIL-6 WT or R167A, E171A and R178E mutants, based on three independent experiments. The data is presented as mean ± SD, n = 3. *p < 0.05; **p < 0.01.

Article Snippet: Subsequently, the cells were washed and stained for 30 min at room temperature in the dark with anti-pSTAT3 (pY705)-PE and an anti-mouse immunoglobulin G isotype-matched control (BD, Cat, 558595).

Techniques: Staining, Negative Control, Flow Cytometry, Labeling

Journal: bioRxiv

Article Title: LDL-Binding IL-10 Reduces Vascular Inflammation in Atherosclerotic Mice

doi: 10.1101/2024.03.04.582839

Figure Lengend Snippet: Fusing IL-10 to an LDL-binding antibody fragment enables lipoprotein binding while maintaining signaling and anti-inflammatory properties. a) Conceptual schematic of plaque-targeted IL-10. b) SDS PAGE gel showing various Fab-IL-10 constructs. N, non-reducing conditions. R, reducing conditions. c-e) Binding affinity of Fab-IL-10 to LDL measured using surface plasmon resonance. KD, dissociation constant. Dashed lines represented calculated fit. f) Activity of IL-10 and Fab-IL-10 by phosphorylation of STAT3 (pSTAT3), measured by flow cytometry of RAW 264.7 cells incubated with the indicated concentrations of IL-10 (n = 3). g) Log(EC50) calculated from fitted curves in (f), shown with 95% confidence intervals. h) TNFα secretion of LPS-stimulated RAW 264.7 cells incubated with WT IL-10 or Fab-IL-10 (n = 5). Experiments were performed twice with similar results. Data represent mean +/-standard deviation (f) or mean + standard deviation (h). Statistics performed by one-way ANOVA with Dunnett’s post-test compared to media.

Article Snippet: Cells were stained with PE-conjugated antibodies against pSTAT3 (pY705, clone 4/P-STAT3, BD Biosciences) at a 1:50 dilution for 1 hr at room temperature in the dark.

Techniques: Binding Assay, SDS Page, Construct, SPR Assay, Activity Assay, Flow Cytometry, Incubation, Standard Deviation

Journal: Cell

Article Title: Human MCTS1-dependent translation of JAK2 is essential for IFN-γ immunity to mycobacteria

doi: 10.1016/j.cell.2023.09.024

Figure Lengend Snippet: Key resources table

Article Snippet: Human pSTAT3-PE (pY705) , BD , Cat# 612569, RRID:AB_399860.

Techniques: Virus, Recombinant, Staining, Selection, Polymerase Chain Reaction, Plasmid Preparation, Reporter Assay, Sequencing, Mutagenesis, Variant Assay, Clone Assay, Knock-Out, Software, Next-Generation Sequencing