rabbit polyclonal anti phospho stat6 antibody  (R&D Systems)

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:

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: Nature metabolism

Article Title: Fasting-induced FOXO4 blunts human CD4 + T helper cell responsiveness

doi: 10.1038/s42255-021-00356-0

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-p-STAT6 (Y641) , Cell Signaling , Cat. #9361S; RRID:AB_331595.

Techniques: Virus, Recombinant, Cell Stimulation, Staining, Flow Cytometry, CyQUANT Assay, Proliferation Assay, Enzyme-linked Immunosorbent Assay, Control, Software, Cell Isolation, In Vitro, Transfection

Journal: eLife

Article Title: Profiling of myristoylation in Toxoplasma gondii reveals an N -myristoylated protein important for host cell penetration

doi: 10.7554/eLife.57861

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-phospho-Stat6 polyclonal , Cell Signaling , Cat# 9361 RRID: AB_331595 , IFA (1:600).

Techniques: Sequencing, Mass Spectrometry, Expressing, Construct, Software