Journal: bioRxiv

Article Title: A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

doi: 10.1101/708503

Figure Lengend Snippet: A. CD163 surface expression in PBS or Kp52145-infected pBMDMs by flow cytometry. Values are shown as standard error of mean of two independent experiments in duplicate. **, p<0.01 determined by unpaired Student’s -test. B. Immunoblotting analysis of phosphorylation of STAT6 (PSTAT6) and tubulin in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. C. cd163 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. D. Arginase-1 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS 1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. E. Immunoblotting analysis of phosphorylation of STAT6 (PSTAT6) and tubulin in lysates of pBMDMs infected with Kp52145 and the isogenic cps mutant, strain 52145-Δ wca K2 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. F. cd163 levels in pBMDMs non-infected (ni) or infected with the cps mutant, strain 52145-Δ wca K2 , pre-treated with STAT6 inhibitor (AS1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments in duplicate. G. arginase-1 levels in pBMDMs non-infected (ni) or infected with cps mutant, strain 52145-Δ wca K2 , pre-treated with STAT6 inhibitor (AS 1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments in duplicate. In panels C, and D, ***p<0.001, **p<0.01, for the indicated comparisons using one-way ANOVA with Bonferroni correction.

Article Snippet: Primary antibodies included: phospho-STAT6 (Tyr641) (1:2000, #9361), total STAT6 (1:1000, BioRad #170-6516), phospho-STAT3 (Y705) (1:2000, #9145), total STAT3 (1:2000, #12640), phospho-ERK (p44/42) (1:2000; #91015), phopsho-p38 (T180/Y182) (1:2000, #4511), all from Cell Signalling Technologies.

Techniques: Expressing, Infection, Flow Cytometry, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

doi: 10.1101/708503

Figure Lengend Snippet: A. il-10 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS 1517499, 50nM/ 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. B. Immunoblotting analysis of phosphorylation of STAT3 (PSTAT3) and STAT3 in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. C. Immunoblotting analysis of phosphorylations of ERK (pERK), p38 (Pp38), and tubulin in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. D. il-10 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with p38 inhibitor (SB203580, Tocris, 10 μg/mL, 2 h prior to infection), ERK inhibitor (U0126, LC laboratories, 20 μg/mL, 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. In panels A and D, ****p < 0.0001, ***p < 0.001, for the indicated comparisons using one-way ANOVA with Bonferroni correction.

Article Snippet: Primary antibodies included: phospho-STAT6 (Tyr641) (1:2000, #9361), total STAT6 (1:1000, BioRad #170-6516), phospho-STAT3 (Y705) (1:2000, #9145), total STAT3 (1:2000, #12640), phospho-ERK (p44/42) (1:2000; #91015), phopsho-p38 (T180/Y182) (1:2000, #4511), all from Cell Signalling Technologies.

Techniques: Infection, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 1. IL-4 dependent Stat6 activation in A201.1 cells is tran- sient when cells are stimulated with IL-4 under pulse condi- tions. A201.1 cells were treated with IL-4 either continuously (A) or pulsed for 15 min (B) and then washed and cultured for the indicated time periods. Cytoplasmic and nuclear extracts were prepared and analyzed for the presence of activated Stat6 by EMSA. In panels C and D, A201.1 cells were pulsed with IL-4 for 15 min, washed, and then cultured for the indicated time periods. Stat6 was precipitated from whole cell extracts (C) or nuclear extracts (D) using anti-Stat6 poly- clonal antibody coupled to protein A/G-agarose. Immunoprecipitates were analyzed by Western blot using the anti-phosphotyrosine antibody 4G10. The blot was stripped and reprobed using an anti-Stat6 poly- clonal antibody.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 2. Novel protein synthesis is not required for the mainte- nance of prolonged Stat6 activation in A201.1 cells. A201.1 cells were cultured in the presence of IL-4 for the time points indicated, either in the absence (A) or presence (B) of cycloheximide (5 g/ml). Cytoplasmic and nuclear extracts were prepared and analyzed for the presence of activated Stat6 by EMSA.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 5. The half-life of Stat6 in A201.1 cells is unaffected by treatment with IL-4. A201.1 cells were incubated in the presence of cycloheximide (5 g/ml) to inhibit protein synthesis. After 1 h, media or IL-4 (10 ng/ml) was added to the cells in the continued presence of cycloheximide and cultured for the indicated time periods. Cells were then lysed, and Stat6 levels were determined by Western blot using an anti-Stat6 polyclonal antibody.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 7. Maintenance of Stat6 activation in A201.1 cells requires nuclear export. A201.1 cells were incubated in the presence of IL-4 (10 ng/ml) for the time periods indicated in the presence or absence of LMB (10 ng/ml). Cells remained 95% viable at all time points at this concentration. Nuclear extracts were made and analyzed for the pres- ence of Stat6 DNA binding activity.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Incubation, Concentration Assay, Binding Assay, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 8. Stat6 becomes localized to the nucleus after 8 h of LMB treatment. A201.1 cells were incubated in the presence of IL-4 (10 ng/ml) for the time periods indicated in the presence or absence of LMB (10 ng/ml). Cells remained 95% viable at all time points at this concentra- tion. Cells were then stained for Stat6 using an anti-Stat6 antibody and analyzed by confocal microscopy. Untreated cells and cells stained with an isotype-matched control antibody are shown for comparison.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, Staining, Confocal Microscopy, Control, Comparison

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 3. Maintenance of Stat6 activation requires ongoing ki- nase activity. A201.1 cells were treated with 10 ng/ml IL-4 for 15 min to achieve maximal Stat6 activation. 500 nM staurosporine or Me2SO (DMSO) vehicle alone were added to the culture media in the continued presence of IL-4 for the time indicated. Nuclear extracts were analyzed for Stat6 DNA binding activity by EMSA.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Activity Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 4. Maintenance of Stat6 activation requires ongoing JAK kinase activity. A201.1 cells were treated with IL-4 to achieve maxi- mal Stat6 activation (15 min). 400 M AG490 (A) or Me2SO (DMSO) vehicle alone (D) were added to the culture media in the continued presence of IL-4 for the time indicated. Cells remained 95% viable after treatment with AG490 at all time points. For comparison, cells were treated with IL-4 for 15 min, washed, and then incubated in media lacking IL-4 for 2 h (lane 3). Nuclear extracts were analyzed for Stat6 DNA binding activity by EMSA.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Activity Assay, Comparison, Incubation, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 6. Prior cycles of Stat6 activation and deactivation do not deplete the pool of functionally available Stat6. A201.1 cells were pulsed with IL-4 for 15 min and then washed and cultured for the indicated time periods. At 2 and 6 h, cells were restimulated with IL-4 for 15 min. Nuclear extracts were prepared and analyzed by EMSA for the presence of activated Stat6.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 9. Sustained Stat6 activation is required to up-regulate IL-4-depend- ent gene expression in A201.1 cells. A201.1 cells were treated with IL-4 either continuously for 48 h or pulsed with IL-4 for 15 min, washed, and cultured for 48 h. A, cells that were treated continuously (left panel) or pulsed (right panel) with IL-4 were analyzed for IL-4-dependent CD23 expression by flow cytometry. B, cytoplasmic and nuclear extracts from cells treated continuously with IL-4 were prepared and analyzed for the presence of activated Stat6 by EMSA. C, a schematic overview of the experimental design is shown at the top. Cells were incubated for a total of 48 h and treated with IL-4 either continuously (upper left) or pulsed, washed, and then cultured in the absence of IL-4 for the remainder of the 48 h pe- riod (upper right). Shaded areas indicate the time in hours that cells were exposed to IL-4. Arrows indicate the points at which IL-4 was added, and the numbers next to the arrows indicate the total amount of time (in hours) the cells were exposed to IL-4. After both treatment con- ditions outlined above, cells were ana- lyzed for CD23 surface expression by flow cytometry at 48 h, which is displayed graphically.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Gene Expression, Cell Culture, Expressing, Flow Cytometry, Incubation

Journal: Journal of Biological Chemistry

Article Title: Analysis of the Life Cycle of Stat6

doi: 10.1074/jbc.m200986200

Figure Lengend Snippet: FIG. 10. Proposed model of Stat6 cycling.

Article Snippet: Polyclonal anti-Stat6 antibody, S-20, was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and anti-phospho STAT6 was purchased from Cell Signaling Technology (Beverly, MA).

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: Opposing control of rhabdomyosarcoma growth and differentiation by myogenin and interleukin 4

doi: 10.1158/1535-7163.mct-08-0678

Figure Lengend Snippet: Figure 4. A, forced expression of myogenin inhibited RD/12 cell migra- tion and blocked directed migration toward IL-4. Columns, mean number of cells migrated in the lower compartment containing medium with or without IL-4, four independent replicates; bars, SE. Migration of RD/ 12-Myog cells was significantly lower than parental RD/12 cells (P < 0.001, Student's t test); IL-4 significantly (stars, P < 0.05, Student's t test) increased migration only of RD/12 and RD/12-Neo cells, but not of RD/12-Myog and RD/18 cells expressing high myogenin levels. IL-4 re- ceptor level (B) and signaling (C) in rhabdomyosarcoma cells with forced myogenin expression. Total proteins (100 μg) were loaded for the evalua- tion of total-STAT6, phospho-STAT6 (STAT6 P), IL-4 Rα, and IL-13 Rα ex- pression. Actin (15 μg of total proteins) was used as a housekeeping protein for sample normalization. M, marker of molecular weight.

Article Snippet: Mouse antihuman IL-13 Rα1 monoclonal antibody (clone 419718, 2 μg/mL), goat anti-human IL-4 Rα antibody (0.2 μg/mL), rabbit anti–phospho-STAT6 (Y641) antibody (0.5 μg/mL; R&D Systems), rabbit anti-total STAT6 (1.5 μg/mL, M-20; Santa Cruz Biotechnology), and rabbit anti-actin (Sigma) were used as primary antibodies.

Techniques: Expressing, Migration, Marker, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

doi: 10.1074/jbc.M110.112359

Figure Lengend Snippet: Deletion of the ROP16 locus identifies STAT6 as a potential target. A, schematic of Δrop16 generation (not to scale). The top line shows the construct used with the Toxoplasma sequences derived from the ROP16 flanks shown as a thick line. Bent arrows indicate promoters driving the negative (GFP) and positive (HXGPRT) selectable markers. The middle line represents the region of chromosome VIIb containing ROP16. The bottom line shows the resulting deletion of the ROP16 locus. Primers used for PCR detection of homologous integration of the targeting construct are indicated by horizontal arrows. B, confirmation of replacement of the ROP16 locus with HXGPRT by PCR. Correct 5′- and 3′-integration was demonstrated with primer pairs 1/2 and 3/4 of Fig. 2A, respectively. Primers 5 and 6 were used to demonstrate the loss of the ROP16 coding region in the Δrop16 mutant. Toxoplasma surface antigen 1 (SAG1) primers were used as a positive control for the PCRs. C and D, microarray analysis of HFFs infected with either WT or Δrop16 (KO) parasites at 1 h (C) and 5 h (D) postinfection. Expression data for HFFs 5 h post-infection with ROP16-complemented Δrop16 parasites or control Δrop16 parasites that underwent transfection and selection alongside the complemented parasites (KO.c) are shown in D. Two biological replicates of each infection per time point were analyzed. Genes shown are those identified as significantly up-regulated by SAM analysis in cells infected with WT versus Δrop16 parasites and >2-fold change in expression (C) or >3-fold change in expression in cells infected with either WT or complemented parasites versus Δrop16 parasites (D). Genes that have previously been characterized as STAT6-regulated are highlighted in gray; genes not known to be regulated by STATs are in white.

Article Snippet: Immunoblots were carried out using the following primary antibodies: mouse monoclonal anti-STAT6 (BD Biosciences), rat monoclonal anti-HA (3F10, Roche Applied Science), mouse monoclonal anti-ROP1 (Tg49) ( 19 ), or mouse polyclonal anti-toxofilin antiserum ( 20 ).

Techniques: Construct, Derivative Assay, Mutagenesis, Positive Control, Microarray, Infection, Expressing, Transfection, Selection

Journal: The Journal of Biological Chemistry

Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

doi: 10.1074/jbc.M110.112359

Figure Lengend Snippet: STAT6 activation is ROP16-dependent and much faster than IL-4-induced activation. A, parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B, HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.

Article Snippet: Immunoblots were carried out using the following primary antibodies: mouse monoclonal anti-STAT6 (BD Biosciences), rat monoclonal anti-HA (3F10, Roche Applied Science), mouse monoclonal anti-ROP1 (Tg49) ( 19 ), or mouse polyclonal anti-toxofilin antiserum ( 20 ).

Techniques: Activation Assay, Infection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

doi: 10.1074/jbc.M110.112359

Figure Lengend Snippet: Tyrosine 641 of STAT6 is directly phosphorylated by ROP16. Recombinant STAT6 (rSTAT6) was incubated with recombinant JAK3 (rJAK3) or recombinant (E. coli-produced) ROP16-HA (WT) or the kinase-deficient K404N (KD) in kinase assay conditions for 30 min at 30 °C, followed by SDS-PAGE and immunoblotting (IB) using antibodies specific for STAT6 phosphorylated on the key activation residue for STAT6, tyrosine 641 (Y641; top), antibodies that recognize STAT6 regardless of activation state (middle), or antibodies for HA-tagged ROP16 (bottom). Molecular masses are noted in kDa on the left.

Article Snippet: Immunoblots were carried out using the following primary antibodies: mouse monoclonal anti-STAT6 (BD Biosciences), rat monoclonal anti-HA (3F10, Roche Applied Science), mouse monoclonal anti-ROP1 (Tg49) ( 19 ), or mouse polyclonal anti-toxofilin antiserum ( 20 ).

Techniques: Recombinant, Incubation, Produced, Kinase Assay, SDS Page, Western Blot, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

doi: 10.1074/jbc.M110.112359

Figure Lengend Snippet: Co-immunoprecipitation of ROP16 and STAT6 in infected cells. HFFs were infected at MOI ∼10 with Type I parasites ectopically expressing HA-tagged wild type ROP16 (WT) or ROP16-K404N (KD). 2 h postinfection, cells were washed with cold PBS and lysed in situ. Immunoprecipitation was carried out on clarified lysate (input) for 12 h at 4 °C using either nonspecific rabbit IgG-conjugated to agarose beads (Control IP) or anti-STAT6 polyclonal rabbit IgG conjugated to agarose beads (STAT6 IP). Immunocomplexes were washed in lysis buffer at 4 °C and then eluted in SDS sample buffer. Input and eluates were analyzed by SDS-PAGE and immunoblotting (IB) using antibodies specific for STAT6 (top panel), HA epitope of ROP16 (second panel), and parasite-secreted proteins ROP1 (third panel) and toxofilin (bottom panel). Cell equivalents are noted (1× or 20×) at the top; molecular masses are noted in kDa on the left.

Article Snippet: Immunoblots were carried out using the following primary antibodies: mouse monoclonal anti-STAT6 (BD Biosciences), rat monoclonal anti-HA (3F10, Roche Applied Science), mouse monoclonal anti-ROP1 (Tg49) ( 19 ), or mouse polyclonal anti-toxofilin antiserum ( 20 ).

Techniques: Immunoprecipitation, Infection, Expressing, In Situ, Lysis, SDS Page, Western Blot

Journal: Neuroreport

Article Title: Cathepsin B promotes optic nerve axonal regeneration

doi: 10.1097/WNR.0000000000002148

Figure Lengend Snippet: Western blot analysis of microglial protein expression following IL-4 and IL-6 activation. (a) Grayscale images depicting the expression levels of STAT3, STAT6, IRE1α, and CTSB in microglia following activation induced by IL-4 and IL-6, as assessed by western blot analysis. (b) The statistical analysis results indicate that, in comparison with the control group, the expression of STAT3, STAT6, IRE1α, and CTSB proteins in microglia is significantly upregulated in the IL-4 + IL-6 group. N = 3, P < 0.05. CTSB, cathepsin B; IL, interleukin; IRE1α, inositol-requiring enzyme 1 alpha; STAT3, signal transducer and activator of transcription 3; STAT6, signal transducer and activator of transcription 6.

Article Snippet: The primar antibodies utilized were: anti-fibronectin (15613-1-AP; Proteintech), antineurocan (1 : 1000; 27087-1-AP, Proteintech), anti-STAT3 (1 : 1000; 10253-2-AP, Proteintech), anti-STAT6 (1 : 1000; 82630-1-RR, Proteintech), anti-IRE1α (1 : 1000; 27528-1-AP, Proteintech), and anti-CTSB (1 : 1000; 12216-1-AP, Proteintech).

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

Journal: Neuroreport

Article Title: Cathepsin B promotes optic nerve axonal regeneration

doi: 10.1097/WNR.0000000000002148

Figure Lengend Snippet: Immunofluorescence analysis of microglial protein expression following IL-4 and IL-6 activation. (a) The expression levels of STAT3, STAT6, IRE1α, and CTSB in microglia following activation induced by IL-4 and IL-6 were assessed using immunofluorescence methods. (b) The statistical analysis results indicate that, in comparison with the control group, the expression of STAT3, STAT6, IRE1α, and CTSB proteins in microglia is significantly upregulated in the IL-4 + IL-6 group. N = 3, P < 0.05, scale bar = 50 μm. CTSB, cathepsin B; IL, interleukin; IRE1α, inositol-requiring enzyme 1 alpha; STAT3, signal transducer and activator of transcription 3; STAT6, signal transducer and activator of transcription 6.

Article Snippet: The primar antibodies utilized were: anti-fibronectin (15613-1-AP; Proteintech), antineurocan (1 : 1000; 27087-1-AP, Proteintech), anti-STAT3 (1 : 1000; 10253-2-AP, Proteintech), anti-STAT6 (1 : 1000; 82630-1-RR, Proteintech), anti-IRE1α (1 : 1000; 27528-1-AP, Proteintech), and anti-CTSB (1 : 1000; 12216-1-AP, Proteintech).

Techniques: Immunofluorescence, Expressing, Activation Assay, Comparison, Control

Journal: The FASEB Journal

Article Title: IL‐13 signaling through IL‐13 receptor α2 mediates airway epithelial wound repair

doi: 10.1096/fj.201801285r

Figure Lengend Snippet: Figure 4. The blocking of IL-13Ra2 via antibody neutralization reduces epithelial repair. Mechanically wounded monolayers of 1HAEo– cells were treated with increasing concentrations of IL-13Ra1 and IL-13Ra2 neutralizing antibodies immediately after injury. 1HAEo– cells in the control group were wounded and maintained in serum-free medium. A) Corresponding wound areas at time 0 and 24 h were used to determine the percentages of repair over 24 h. The percentage of repair at 24 h postinjury was significantly reduced in the IL-13Ra2 neutralizing antibody (4 mg/ml) treatment group compared with the serum-free media control group. *P , 0.05 vs. serum-free media control, n = 4, 1-way ANOVA with Bonferroni multiple comparison test. B) Images represent mechanically wounded monolayer cultures of 1HAEo– cells treated with IL-13Ra1 or IL-13Ra2 neutralizing antibody. Cultures were imaged at 0, 6, and 24 h postinjury to assess repair. C) Pretreatment with IL-13Ra1 neutralizing antibody before IL- 13 exposure reduced STAT6 activation compared with the time-matched control at 2 h post-treatment in 1HAEo– cells. **P , 0.01, n = 3, 2-way ANOVA with Bonferroni posttest. D) 1HAEo– cells that were pretreated with IL-13Ra1 neutralizing antibody have reduced MUC5AC transcript expression, 4 and 6 h post-IL-13 exposure, as measured by qPCR. ***P , 0.001, *P , 0.05 vs. control, n = 3, 2-way ANOVA with Bonferroni posttest.

Article Snippet: Membranes were incubated with the following primary antibodies overnight at 4°C: rabbit anti-human IL-13Ra1 antibody (ab79277; 1mg/ml; Abcam, Cambridge,MA, USA), goat anti-human IL-13Ra2 antibody (0.2 mg/ml; R&D Systems), rabbit anti-phosphorylated EGFR (p-EGFR) antibody (44784G; 1 mg/ml; Thermo Fisher Scientific), sheep anti-EGFR antibody (20-ES04; 1mg/ml; Fitzgerald,NorthActon,MA,USA), goat anti-HB-EGF antibody (sc-1413; 1 mg/ml; Santa Cruz Biotechnology,Dallas, TX,USA), rabbit anti-phosphorylated STAT6 (p-STAT6) antibody (9361; 0.1 mg/ml; Cell Signaling Technology, Danvers, MA, USA), and mouse anti-STAT6 antibody (611,290; 0.25 mg/ml; BD BioSciences, Mississauga, ON, Canada).

Techniques: Blocking Assay, Neutralization, Control, Comparison, Activation Assay, Expressing

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