ser9 5b3 cell signaling technology 9323p rrid ab 2115201 rabbit  (Cell Signaling Technology Inc)

Journal: Molecular immunology

Article Title: Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization.

doi: 10.1016/j.molimm.2022.11.002

Figure Lengend Snippet: Fig. 6. Phosphorylation of STAT3 and STAT6 proteins in rat macrophages. pSTAT3/STAT3 ratio (a) and protein bands (b., pSTAT3, STAT3 and GAPDH) are shown. JAK/ STAT kinase inhibitors and IL-4 were added to macro phages from casein-treated rats during 24 h culture. IL-4 treatment caused significant (*p < 0.05), while AT and CYT caused strongly significant (***p < 0.001) decrease in phosphorylation of STAT3. Polyclonal anti-pSTAT3 anti body (rabbit, R & D Systems, 1:1000), monoclonal anti- STAT3 antibody (mouse, R & D Systems, 1:2000) and anti-GAPDH (R& D Systems, 1:3000) were used as primary, while HRPO-conjugated goat anti-rabbit (R & D Systems, 1:2000) and goat anti-mouse (R & D Systems, 1:3000) were used as secondary antibodies. c.) Results of cell-based ELISA experiments for pSTAT6 and STAT6 are shown. * *p < 0.01 between res 0′ and cas 0′; §p < 0.05 between casein 0′ and 24 h; ###p < 0.001 for IL-4, AT and CYT treatments compared to casein 24 h as control. Polyclonal anti-pSTAT6 antibody (rabbit, R & D Systems, 1:200), monoclonal anti-STAT6 antibody (mouse, R & D Systems, 1:200) and monoclonal anti-GAPDH (mouse, R & D Sys tems, 1:300) were used as primary, while HRPO- conjugated goat anti-rabbit (R & D Systems, 1:800) and goat anti-mouse (R & D Systems, 1:800) were used as secondary antibodies.

Article Snippet: Fixed cells were then treated with 50 μl rabbit polyclonal anti-phospho-STAT6 antibody (1:200, R & D Systems, MN USA), 50 μl mouse monoclonal anti-STAT6 antibody (1:200, R & D Systems) and 50 μl mouse monoclonal anti-GAPDH antibody (1:300, R & D Systems) for overnight at 4 ◦C.

Techniques: Phospho-proteomics, In-Cell ELISA, Control

Journal: Journal of pharmacological sciences

Article Title: Seihaito, a Kampo medicine, attenuates IL-13-induced mucus production and goblet cell metaplasia.

doi: 10.1016/j.jphs.2024.02.008

Figure Lengend Snippet: Fig. 3. Effect of Seihaito on IL-13-induced STAT6 phosphorylation and SPDEF mRNA expression in ALI-cultured tracheal epithelial cells. ALI cultures of tracheal epithelial cells were treated with IL-13 (10 ng/ml) combined with Seihaito (1 mg/ml) or DEX (100 nM) for 7 days. Phosphorylation of STAT6 was assessed by western blotting (A), and SPDEF mRNA was measured by RT-real-time PCR (B). Data are presented as mean ± SE from three different experiments.

Article Snippet: Rabbit polyclonal anti-STAT6 antibody and rabbit monoclonal antiphospho-STAT6 antibody (Y641), mouse anti-β-actin antibody were purchased from Cell Signaling (MA, USA).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Transcriptional landscape of the dorsal raphe serotonin neurons rendering stress resiliency

doi: 10.1101/2024.03.21.586199

Figure Lengend Snippet: (a) IPA comparison analysis of upstream pathways. (b) A schematic illustration of the IL4-STAT6 signaling pathway. (c) Immunofluorescence image (left) and quantification of S100a10 (right) in DRN of SSRI-treated mice at each time points. Scale bar, 200 μm (top pannel), 20 μm (middle and bottom panels). n = 9-10. **P < 0.01, compared to 0 day, one-way ANOVA, Dunnett’s post hoc test. (d) Immunofluorescence image (left) and quantification of pSTAT6 (phosphor Y641) (right) in DRN of SSRI-treated mice at each time point. Scale bar, 20 μm. n = 9-10. *P < 0.01, compared to 0 day, one-way ANOVA, Dunnett’s post hoc test. (e) ELISA measurements for IL-4 (left) and IL-13 (middle) levels of DRN at each time point after SSRI treatment. Total protein levels of DRN samples measured by BCA assay (right). n = 9-10. Data presented as means ± s.e.m. n = 9-10. *P < 0.01, compared to 0 day, one-way ANOVA, Dunnett’s post hoc test. (f) An illustration of the STAT6 mutant used in the following experiments. (g) Luciferase activity of 48 h posttransfection of hSTAT6 YF or pcDNA in either unstimulated HepG2 cells or cells that had been treated with IL-4 6 h prior to harvest. We conducted two independent assays (n = 3 each) and the luciferase activity was normalized by each batch of the IL-4 (+) YF (-) group. ****P < 0.0001, compared to IL-4 (-) YF (-), ####P < 0.0001, compared to IL-4 (+) YF (-), respectively by One-way ANOVA, Tukeys post hoc test. (h) Schematic representation of AAV injection and time course of experiments. (i) Total distance in OFT, (j) time spent in open arm in EPM, (k) total immobility time in TST. n = 14-15. ****P < 0.0001, two-tailed t-test. (l) Schematic representation of drug injection and time course of experiments. (m) Total distance in OFT, (n) time spent in open arm in EPM, (o) total immobility time in TST. n = 11-12. ***P < 0.001, compared to saline, one-way ANOVA, Dunnett’s post hoc test. (p) Immunofluorescence image (top) and quantification of S100a10 (bottom) in DRN of IL4-injected mice at each time points. Scale bar, 20 μm. n = 11-12. *P < 0.05, compared to saline, one-way ANOVA, Dunnett’s post hoc test. All data presented as means ± s.e.m.

Article Snippet: Then sections were incubated overnight at 4 ◦C with goat polyclonal anti-S100a10 antibody (1:200; AF2377, R&D Systems, Minneapolis, MN, USA), rabbit polyclonal anti-5HT 1B Receptor antibody (1:100; ab13896, abcam, Cambridge, UK), sheep polyclonal anti-tryptophan hydroxylase (TPH) antibody (1:500; AB1541, Merck Millipore, Burlington, MA, USA), and rabbit polyclonal Anti-STAT6 (phospho Y641) antibody (1:100; ab28829, abcam) diluted in the blocking buffer.

Techniques: Comparison, Immunofluorescence, Enzyme-linked Immunosorbent Assay, BIA-KA, Mutagenesis, Luciferase, Activity Assay, Injection, Two Tailed Test, Saline

Journal: Frontiers in Immunology

Article Title: Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6

doi: 10.3389/fimmu.2023.1289795

Figure Lengend Snippet: MVP interacts with STAT6 and JAK1. (A) HA-tagged MVP and Flag-tagged STAT6 were co-transfected into HEK293T at 36 h post-transfection, and the cells were collected for Co-immunoprecipitation (Co-IP) and immunoblot analyses. (B) BMDMs were cultured in SF medium overnight after the formation of adherent cells. The next day, cells were stimulated with medium or IL-4 for 30 min and collected for Co-IP and immunoblot analyses. (C) HEK293T cells were transfected with pEGFP-MVP and DsRed-STAT6 for 36 h and then collected for IF assays. Representative image of MVP (green) and STAT6 (red), Scale bar, 10 µm (left panel). The quantitative analysis of co-localization using Image J (right panel). (D) Schematic diagram of structural domains and truncated constructs of STAT6 full-length (FL) (upper panel). HEK293T were co-transfected with HA-tagged MVP and truncated mutants of Flag-tagged STAT6 for 36 h. Then cells were collected for Co-IP and immunoblot analyses (lower panel). (E) Experiments were performed similarly to those in (D) , except the indicated truncated constructs of MVP were used. (F) Flag-JAK1 and HA-MVP were co-transfected into HEK293T for 36 h, and then the cells were harvested for Co-IP and immunoblot analyses. (G) BMDMs were stimulated with or without IL-4 for 30 min and collected for Co-IP and immunoblot analyses. (H) HEK293T cells were transfected with indicated plasmids for 36 h prior to Co-IP and immunoblot analyses. (I) Wt and Mvp -/- BMDMs were stimulated with medium or IL-4 for 30 min prior to Co-IP and immunoblot analyses. All protein abundance analyses were performed using Image J. All experiments were repeated at least three times with similar results. See also Supplementary Figure 7 .

Article Snippet: Antibodies for phospho-STAT6 (Tyr641) (CSB-PA000625) and phospho-STAT1 (Tyr641) (CSB-PA050162) were from Cusabio.

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Construct, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6

doi: 10.3389/fimmu.2023.1289795

Figure Lengend Snippet: MVP enhances the phosphorylation and nuclear translocation of STAT6. (A) Wt and Mvp -/- BMDMs were stimulated with or without IL-4 at the indicated times before western blot analysis. (B) Raw264.7 cells were transfected with Flag-tagged MVP or vector for 36 h, then stimulated with phosphate-buffered saline (PBS) or IL-4 for 30 min. Immunoblot analyses were performed with the indicated antibodies. (C) Wt and Mvp -/- BMDMs were stimulated with IL-4 for the indicated time. The whole cell lysates (WCL), cytosolic and nuclear extracts were prepared and subjected to western blot analyses. Lamin B and GAPDH were used as nuclear and cytosolic fractions markers, respectively. (D) Experiments were performed similar to those in (C) , except Raw264.7 cells were transfected with Flag-MVP for 36 h. (E) IF of STAT6 in Wt and Mvp -/- PMs stimulated with IL-4. Representative image of STAT6 (green). Scale bar, 20 µm (left panel). The percentage of nuclear STAT6 positive cell numbers was counted (right panel). All protein abundance analyses were performed using Image J. All experiments were repeated at least three times with similar results. Data are expressed as means ± SEM, n = 3, two-tailed Student’s t-test. (**P < 0.01). See also Supplementary Figure 8 .

Article Snippet: Antibodies for phospho-STAT6 (Tyr641) (CSB-PA000625) and phospho-STAT1 (Tyr641) (CSB-PA050162) were from Cusabio.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Transfection, Plasmid Preparation, Saline, Quantitative Proteomics, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Major vault protein regulates tumor-associated macrophage polarization through interaction with signal transducer and activator of transcription 6

doi: 10.3389/fimmu.2023.1289795

Figure Lengend Snippet: MVP-promoted M2-TAMs polarization and tumorigenesis in HCC. In the tumor microenvironment of hepatocellular carcinoma, JAK1 recruits MVP and STAT6, leading to ternary complex formation. Then, STAT6 is phosphorylated and translocated from the cytosol to the nucleus. As a result, STAT6 binds to the promoter of M2 genes, leading to M2 polarization and M2-TAMs infiltration.

Article Snippet: Antibodies for phospho-STAT6 (Tyr641) (CSB-PA000625) and phospho-STAT1 (Tyr641) (CSB-PA050162) were from Cusabio.

Techniques: