mouse phospho stat6  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Medicine

Article Title: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2

doi: 10.3892/ijmm.2026.5764

Figure Lengend Snippet: HA from myofibroblasts induces macrophage M2 polarization via the CD44/STAT6 axis. (A) RAW264.7 macrophages were stimulated with HA for 24 h, and flow cytometry was performed to assess the expression of CD206 (FITC) and CD86 (PE) (n=3 for each group). (B) The content of TGF-β1 in macrophages following HA treatment over 48 h was quantified using ELISA (n=3 for each group). (C) Western blotting analysis of p-STAT6 and total STAT6 expression at various time points following 100 μ g/ml HA stimulation (n=4 for each group). (D) Flow cytometric analysis of CD86 and CD206 expression in macrophages after 24-h treatment with 100 μ g/ml HA and CD44 inhibitor at indicated concentrations (n=3 for each group). (E) TGF-β1 quantification by ELISA in culture supernatants (n=3 for each group). (F) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and CD44 inhibitor at varying concentrations (n=3 for each group). (G) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and STAT6 inhibitor (AS1517499) at varying concentrations (n=4 for each group). (H) Schematic workflow: Conditioned medium from TGF-β1-stimulated NIH/3T3 fibroblasts was used to culture RAW264.7 macrophages for 24 h. Where indicated, RAW264.7 cells were co-treated with a CD44 inhibitor or a STAT6 inhibitor. Macrophage polarization was subsequently analyzed by flow cytometry. (I) Flow cytometry was used to measure the expression levels of CD206 (FITC) and CD86 (PE) (n=3 for each group). * P<0.05, ** P<0.01, *** P<0.001. HA, hyaluronic acid; FITC, fluorescein isothiocyanate; PE, phycoerythrin; p-, phosphorylated.

Article Snippet: Antibodies for Collagen type I α 1 chain (COL1A1; cat. no. 72026; 1:1,000), Phospho-STAT6 (Tyr641; cat. no. 56554S; 1:1,000) and α-smooth muscle actin (α-SMA; cat. no. 19245; 1:1,000) were obtained from Cell Signaling Technology, Inc.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Immunoblots of pSTAT6 (pY641) and γH2AX in thioglycolate-elicited peritoneal macrophages (Thio-PM) with Etoposide (Eto) and IL-4 treatment. The right panel shows the relative intensity of pSTAT6 (pY641) ( n = 4 independent experiments). B Immunofluorescence images of staining (pSTAT6 (pY641), red; DAPI, blue) of IL-4-stimulated Thio-PMs treated with Eto. Scale bars 50 μm. Right panel shows the quantification of pSTAT6 (pY641) ( n = 3 biological replicates per group). C Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Thio-PMs with different inhibitors. KU55933, an inhibitor of ATM. Berzosertib, inhibitor of ATR. NU7026, inhibitor of DNA-PK. AZD7762, inhibitor of CHK1/2. The right panel shows the quantification of pSTAT6 (pY641) ( n = 3 independent experiments). D Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with indicated sgRNA. The right panel shows the quantification of pSTAT6 (pY641)/tSTAT6 ( n = 3 independent experiments). E Dual luciferase reporter of STAT6 in RAW 264.7 cells treated with Eto and NU7026 ( n = 3 per group). Data are mean ± s.e.m. p-value was calculated by paired two-tailed Student’s t test ( A , D ), unpaired two-tailed Student’s t test ( B ), one-way ANOVA with Dunnett’s correction ( C , E ).

Article Snippet: Antibodies used were as follows: HSP90 (Santa Cruz, #sc-13119, 1:10,000), α-Tubulin (Sigma, #T6199, 1:10,000), STAT6 (Cell Signaling, #5397, 1:1500), pSTAT6(pY641) (Cell Signaling, #56554, 1:1000), pSTAT6(pS807) (ABclonal Tech, customized, 1:200), DNA-PK (Santa Cruz, #sc-390849, 1:500), ATM (Cell Signaling, #2873, 1:1000), ATR (Santa Cruz, #sc-515173, 1:500), CHK1 (Cell Signaling, #2360, 1:1500), CHK2 (Proteintech, #13954-1-AP, 1:1000), p16-INK4A (Abcam, #ab211542, 1:1000), γH2AX (Cell Signaling, #9718, 1:1500), BRCA1 (Thermo Fisher, #MA1-23164, 1:1000), UBE2T (Thermo Fisher, # PA576202 , 1:1000), JAK (Cell Signaling, #50996, 1:1000), STING (Cell Signaling, #13647, 1:2000), STING (pS365) (Cell Signaling, #72971, 1:1000), pTBK1 (pS172) (Cell Signaling, #5483, 1:1000), pJAK1 (pY1034/1035) (Cell Signaling, #74129, 1:1000), Ubiquitin (Cell Signaling, #3936, 1:1000), K48-specific ubiquitin (Abcam, #ab140601, 1:1000), PU.1 (Abcam, #ab227835, 1:1500), Col1a1 (Cell Signaling, #72026, 1:1000), SMAD2/3 (Cell Signaling, #8685, 1:1000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (Cell Signaling, #8828, 1:1000), αSMA (Cell Signaling, #19245, 1:2000) anti-HA (Cell Signaling, #3724, 1:5000) and anti-Flag (Cell Signaling, #8146, 1:5000).

Techniques: Western Blot, Immunofluorescence, Staining, Luciferase, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Co-IP of STAT6-Turbo and JAK1, DNA-PK, ATM, and ATR in RAW 264.7 cells with TurboID. B Proximity ligation assay (PLA) of STAT6 and DNA-PK in Thio-PMs treated with Eto. Scale bars 10 μm. C The quantification of PLA signal in ( B ) ( n = 3 biological replicates per group, with more than 100 cells). D Diagram of phosphorylated sites in mouse STAT6 after Eto treatment. E Immunoblots of pSTAT6 (pY641) and tSTAT6 in Stat6 -/- Thio-PMs expressing STAT6 mutants (WT, S807A, S807E). F Immunoblots of pSTAT6 (pY641) in STAT6-mutants-expressed Cas9 tg+ Thio-PMs with Prkdc sgRNA under IL-4 & Eto co-treatment. G Dual luciferase reporter of STAT6 in RAW 264.7 cells expressing STAT6 mutants under IL-4 treatment ( n = 3 biological replicates per group). H Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Cas9 tg+ Thio-PMs with Prkdc sgRNAs. I Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with Ku70 or Ku80 sgRNA. J Immunoblots of pSTAT6 (pS807) and γH2AX in Eto-treated Thio-PMs pre-treated with NU7026. The right panel shows the relative intensity of pSTAT6 (pS807) ( n = 3 independent experiments). K Immunoblots of pSTAT6 (pS807) and pSTAT6 (pY641) in Thio-PMs with indicated treatment. AS1517499, an inhibitor of STAT6 (block of pY641). L Immunoblots of pSTAT6 (pS807) and tSTAT6 in Eto-treated Stat6 -/- Thio-PMs expressing STAT6 mutants (WT, Y641F). The blot results are representative of three biologically independent repeats in ( A , E , F , H , I , K and L ). Data are mean ± s.e.m. Unpaired two-tailed Welch’s t test ( C ), One-way ANOVA with Dunnett’s correction ( G ), paired two-tailed Student’s t test ( J ).

Article Snippet: Antibodies used were as follows: HSP90 (Santa Cruz, #sc-13119, 1:10,000), α-Tubulin (Sigma, #T6199, 1:10,000), STAT6 (Cell Signaling, #5397, 1:1500), pSTAT6(pY641) (Cell Signaling, #56554, 1:1000), pSTAT6(pS807) (ABclonal Tech, customized, 1:200), DNA-PK (Santa Cruz, #sc-390849, 1:500), ATM (Cell Signaling, #2873, 1:1000), ATR (Santa Cruz, #sc-515173, 1:500), CHK1 (Cell Signaling, #2360, 1:1500), CHK2 (Proteintech, #13954-1-AP, 1:1000), p16-INK4A (Abcam, #ab211542, 1:1000), γH2AX (Cell Signaling, #9718, 1:1500), BRCA1 (Thermo Fisher, #MA1-23164, 1:1000), UBE2T (Thermo Fisher, # PA576202 , 1:1000), JAK (Cell Signaling, #50996, 1:1000), STING (Cell Signaling, #13647, 1:2000), STING (pS365) (Cell Signaling, #72971, 1:1000), pTBK1 (pS172) (Cell Signaling, #5483, 1:1000), pJAK1 (pY1034/1035) (Cell Signaling, #74129, 1:1000), Ubiquitin (Cell Signaling, #3936, 1:1000), K48-specific ubiquitin (Abcam, #ab140601, 1:1000), PU.1 (Abcam, #ab227835, 1:1500), Col1a1 (Cell Signaling, #72026, 1:1000), SMAD2/3 (Cell Signaling, #8685, 1:1000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (Cell Signaling, #8828, 1:1000), αSMA (Cell Signaling, #19245, 1:2000) anti-HA (Cell Signaling, #3724, 1:5000) and anti-Flag (Cell Signaling, #8146, 1:5000).

Techniques: Co-Immunoprecipitation Assay, Proximity Ligation Assay, Western Blot, Expressing, Luciferase, Blocking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Hypothetical model of regulation of pSTAT6 (pY641). B Immunoblots of pSTAT6 (pY641) and tSTAT6 in STAT6-mutants (WT, S807E)-expressed RAW 264.7 cells under IL-4 stimulation for 30 min or 4 h. C Quantification of pSTAT6 (pY641)/tSTAT6 in ( B ) ( n = 3 independent experiments). D Stabilization assay of STAT6 mutants (WT, S807E) in IL-4-stimulated RAW 264.7 cells under CHX treatment. E Degradation curve of pSTAT6 (pY641) in ( D ) ( n = 3 independent experiments). F Immunoblots of pSTAT6 (pY641) and tSTAT6 in MG132-pre-treated Stat6 -/- PMs expressing STAT6 mutants (WT, S807E) under IL-4 stimulation for 4 h. G Immunoblots of ubiquitination and K48-ubiquitination on STAT6 in Thio-PMs with indicated treatments. The bottom right panel shows the quantification of STAT6 ubiquitination ( n = 3 independent experiments). H Co-IP of RNF128-FLAG with STAT6-HA mutants (WT, S807E) in RAW 264.7 cells after 4-h IL-4 stimulation. I The quantification of the interaction between STAT6 and RNF128 in ( H ) ( n = 4 independent experiments). J Immunoblots of ubiquitination on STAT6-HA mutants (WT, S807E) and their interaction with RNF128-FLAG in RAW 264.7 cells after 4 h IL-4 stimulation. K Immunoblots of ubiquitination on STAT6-HA mutants (WT, K319R, K595R, K319R/K595R) in RAW 264.7 cells after 4-h IL-4 stimulation. L Stability assay of STAT6 mutants (WT, K319R, K595R) in RAW 264.7 cells under CHX treatment. M Stability assay of STAT6 mutants (WT, S807A, S807A/K319R) in RAW 264.7 cells under CHX treatment. N Diagram of pS807 effect on activated STAT6 stability. The blot data are representative of three independent repeats ( F and J – M ). Data are mean ± s.e.m. Paired two-tailed t test ( C , I ), ordinary two-way ANOVA ( E ), one-way ANOVA with Dunnett’s correction ( G ).

Article Snippet: Antibodies used were as follows: HSP90 (Santa Cruz, #sc-13119, 1:10,000), α-Tubulin (Sigma, #T6199, 1:10,000), STAT6 (Cell Signaling, #5397, 1:1500), pSTAT6(pY641) (Cell Signaling, #56554, 1:1000), pSTAT6(pS807) (ABclonal Tech, customized, 1:200), DNA-PK (Santa Cruz, #sc-390849, 1:500), ATM (Cell Signaling, #2873, 1:1000), ATR (Santa Cruz, #sc-515173, 1:500), CHK1 (Cell Signaling, #2360, 1:1500), CHK2 (Proteintech, #13954-1-AP, 1:1000), p16-INK4A (Abcam, #ab211542, 1:1000), γH2AX (Cell Signaling, #9718, 1:1500), BRCA1 (Thermo Fisher, #MA1-23164, 1:1000), UBE2T (Thermo Fisher, # PA576202 , 1:1000), JAK (Cell Signaling, #50996, 1:1000), STING (Cell Signaling, #13647, 1:2000), STING (pS365) (Cell Signaling, #72971, 1:1000), pTBK1 (pS172) (Cell Signaling, #5483, 1:1000), pJAK1 (pY1034/1035) (Cell Signaling, #74129, 1:1000), Ubiquitin (Cell Signaling, #3936, 1:1000), K48-specific ubiquitin (Abcam, #ab140601, 1:1000), PU.1 (Abcam, #ab227835, 1:1500), Col1a1 (Cell Signaling, #72026, 1:1000), SMAD2/3 (Cell Signaling, #8685, 1:1000), p-SMAD2 (Ser465/467)/SMAD3 (Ser423/425) (Cell Signaling, #8828, 1:1000), αSMA (Cell Signaling, #19245, 1:2000) anti-HA (Cell Signaling, #3724, 1:5000) and anti-Flag (Cell Signaling, #8146, 1:5000).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Stability Assay, Two Tailed Test