Journal: Plants

Article Title: Cheungsam Seed Husk Extract Reduces Skin Inflammation through Regulation of Inflammatory Mediator in TNF-α/IFN-γ-Induced HaCaT Cells

doi: 10.3390/plants13121704

Figure Lengend Snippet: Th2 cell suppressive effect of CSSH through JAK1/STAT6 pathway control. HaCaT cells were pre-treated with different concentrations of CSSH extract (25, 50, 100 and 200 ng/mL) and then stimulated with TNF-α/IFN-γ. ( A ) The protein levels of total and phosphorylated forms of JAK1/STAT6 were measured using Western blot analysis. ( B , C ) The band intensity of JAK1/STAT6. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc comparison test between different groups. Data represent as mean ± SD of the three independent experiments. ### p < 0.001 vs. control group; ** p < 0.01 and *** p < 0.001 vs. TNF-α/IFN-γ stimulated group.

Article Snippet: In addition, NLRP3, phospho-ERK1/2, ERK1/2, phospho-JNK, JNK, phospho-p38, p38, phospho-p65, p65, phospho-IκBα, IκBα, α-tubulin, Lamin B1, phospho-JAK1, JAK1, phospho-STAT6, STAT6 and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Control, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Akt, IL-4, and STAT Proteins Play Distinct Roles in Prostaglandin Production in Human Follicular Dendritic Cell-like Cells

doi: 10.3390/ijms242316692

Figure Lengend Snippet: The effect of STAT protein knockdown on the suppressive activity of IL-4. ( A – F ) Indicated STAT proteins or control siRNA-transfected FDC-like cells were cultured in the presence or absence of IL-4 (100 U/mL) for 24 h and then with IL-1β (25 pg/mL) for 4 h. Expression levels of indicated proteins were measured with immunoblotting. Representative immunoblots and statistical analysis data (mean ± SEM) from three independent experiments are shown. The successful knockdown of Akt was verified with immunoblotting. Molecular weights of STATs: STAT1 (91 kDa), STAT2 (113 kDa), STAT3 (86 kDa), STAT4 (81 kDa), STAT5 (90 kDa), and STAT6 (110 kDa). Statistical significance was analyzed with Student’s t -test by comparing with the control (* p < 0.05; ** p < 0.01; *** p < 0.001; ns, nonsignificant).

Article Snippet: Immunoblotting to detect COX-2 (#12282S), total Akt (#9272) and phosphorylated Akt (Ser473, #9271), STAT1 (#9172), STAT2 (#4594), STAT3 (#9132), STAT4 (#2653), STAT5 (#9363), and STAT6 (#9362) proteins was carried out using antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Knockdown, Activity Assay, Control, Transfection, Cell Culture, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Akt, IL-4, and STAT Proteins Play Distinct Roles in Prostaglandin Production in Human Follicular Dendritic Cell-like Cells

doi: 10.3390/ijms242316692

Figure Lengend Snippet: A proposed model for the regulation of PG production in FDC. PGs are produced in FDC during cellular interactions with B cells or in response to inflammatory cytokines, including IL-1β. The upstream enzyme of PGs, COX-2, is increased in FDC after B cell stimulation and IL-1β triggering. Akt plays inhibitory roles during these processes by restraining COX-2 expression. IL-4 also suppresses PG production by repressing COX-2 expression in a STAT6-dependent manner.

Article Snippet: Immunoblotting to detect COX-2 (#12282S), total Akt (#9272) and phosphorylated Akt (Ser473, #9271), STAT1 (#9172), STAT2 (#4594), STAT3 (#9132), STAT4 (#2653), STAT5 (#9363), and STAT6 (#9362) proteins was carried out using antibodies from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Produced, Cell Stimulation, Expressing

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: PARP-1 influences STAT6 protein level and regulates its occupancy on the gata-3 promoter following IL-4 stimulation. A WT and PARP-1 −/− splenocytes isolated from C57BL/6 J mice were stimulated with IL-4 for the indicated time points. Protein lysate were analyzed for STAT6, pSTAT6 (Y641), PARP-1 and Actin. The bands of STAT6 were quantified and expressed as percent change compared with their respective untreated control/actin (right panel). B IL-4-treated cells and relative controls undergo subcellular fractionation and then protein extracts were analyzed for STAT6, Lamin B and Actin. C WT and PARP-1 −/− splenocytes isolated from C57BL/6 J mice were stimulated with IL-4 for the indicated intervals of time. ChIP for STAT6 was performed and immunoprecipitated DNA was analyzed using qPCR targeting the proximal or distal promoter of gata-3. Data are representative of at least three independent experiments. *, # Significant difference from respective controls or experimental sample, respectively ( p < 0.05)

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Isolation, Fractionation, Immunoprecipitation

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: STAT6 interacts with and is PARylated by PARP-1 in a cell-free system with recombinant proteins and in IL-4 stimulated mouse splenocytes. A Workflow of the cell-free PARylation reaction. B Ni–NTA pull down for hSTAT6 was performed and samples were examined with antibodies against PAR, or STAT6. C IL-4-treated splenocytes and controls were collected at the indicated time points. Protein extracts were subjected to immunoprecipitation (IP) with antibodies to STAT6. Subsequently, an immunoblot analysis of the IP material or input proteins was performed using antibodies against STAT6, p-STAT6 (Y641), PAR, PARP-1, or Actin. Data are representative of at least 3 independent experiments

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Recombinant, Immunoprecipitation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: STAT6 is selectively degraded by Calpain-1 but its PARylation protects it against this process . A Recombinant poly-His-STAT6 was phosphorylated in vitro by active JAK3 or left unphosphorylated prior to incubation with calpain-1 for 10 min. The reaction was stopped with sample buffer and analyzed by immunoblot analysis. The bands were quantified and expressed as percent change compared with their respective untreated control (bottom panel). B Calpain-1 was incubated with STAT6, JAK3, JAK1, PARP-1, or PARG for 40 min. Immunoblot analyses were performed with antibodies against STAT6, PARP-1, JAK1, JAK3, or PARG. C Calpastatin was added to the cell free reaction mix containing STAT6 and Calpain-1. Samples were then subjected to immunoblot analysis with antibodies to the respective proteins. D JAK3-mediated STAT6 phosphorylation, PARylation, and calpain enzymatic reactions were performed in this order. Immediately after the phosphorylation the reaction was divided in 4 parts.Then, immunoblot analysis was carried out with antibodies against STAT6, p-STAT6 (Y641), PARP-1, or PAR. E Recombinant STAT6 was incubated with JAK3 in the presence of calpain-1 with or without NAD + . STAT6 degradation was assessed by immunoblot analysis

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Recombinant, In Vitro, Incubation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: STAT6 is degraded by AA starvation-induced autophagy . A – B Jurkat, MEF and PM1 cells were exposed to AA starvation media for the indicated time and then processed for immunoblot analysis. Antibodies against STAT6, LC3, actin, or tubulin were used. C Jurkat cells were cultured for 6 h in starvation media and then supplemented with AA for the indicate times. Protein extracts were subjected to immunoblot analysis with antibodies to STAT6, tubulin, or LC3. D The blot from ( A ) was re-probed with antibodies to STAT1, STAT2, STAT3, STAT4 or PARP-1. E CAPNS1 −/− cells expressing human CAPNS1 or empty vector were subjected to AA starvation for the indicated times. Protein extracts were then subjected to immunoblot analysis with antibodies to STAT6, CAPNS1, LC3 or actin. F Putative CMA-targetable motifs on STAT6. G Jurkat cells subjected to AA starvation for 6 h were concomitantly treated with CMA inducers or inhibitors or calpastatin. Levels of STAT6 and autophagy status were assessed by immunoblot analysis of protein extracts with antibodies to STAT6, LC3 or actin

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Western Blot, Cell Culture, Expressing, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: IL-4 treatment protects STAT6 from autophagy-induced degradation and PARP-1 inhibition abrogates such protection without affecting autophagy . A Splenocytes isolated from WT C57BL/6 J mice were stimulated with IL-4 in the absence or presence of olaparib for the indicated time points. Protein lysates were subjected to immunoblot analysis with antibodies to STAT6 or Actin. B Jurkat cells were exposed to AA starvation media then supplemented with different percentages of AAs in the presence or absence of IL-4 or the PARP inhibitor, olaparib, for 12 h. Protein extracts were then subjected to immunoblot analysis with antibodies to STAT6, p62 or Tubulin. The brackets on the left indicate that the two sets of panels were of same samples but run on two different gels. C Jurkat were cultured in starvation media then supplemented with 25% AAs and treated for 12 h with IL-4, olaparib, calpastatin or combinations of the different agents. Not starved cells were used as control. Protein extracts were then subjected to immunoblot analyses with antibodies to STAT6, STAT4, p62 or actin. D The intensity of the STAT6 bands showed on ( B ) was quantified using ImageJ-Fiji and results were normalized to respective Actin band intensity

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Inhibition, Isolation, Western Blot, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: Synthetic degradation of IL-4-activated STAT6 upon PARP inhibition and its association with calpains and autophagy. IL-4 binds to its receptor (upon the dimerization of the IL-R4α and γC subunits) leading to the recruitment and subsequent activation of JAK1/3 kinases, which culminates in the phosphorylation of STAT6. Phosphorylated STAT6 monomers then dimerize and translocate to the nucleus where they occupy the gata-3 gene promoter. During the early stages after IL-4 stimulation, PARP-1 expression is required for a persistent occupancy of the gata-3 promoter by the STAT6 dimer. Whether PARylation is required here is not clear. At a later stage, phosphorylated STAT6 is PARylated, which protects it from calpain-mediated degradation. Inhibition of PARylation by PARP inhibitors renders STAT6 susceptible to degradation. Autophagy also promotes degradation of STAT6 and is protected by IL-4 stimulation and subsequent PARylation. CMA appears to be the major mechanism by which STAT6 is degraded during autophagy. The increased susceptibility of phosphorylated STAT6 for degradation by calpains upon PARP inhibition may be considered as artificial or synthetic, hence our proposal of naming this process “ synthetic protein degradation ”

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Inhibition, Activation Assay, Expressing