Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Primer list

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Sequencing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Analyses of Aqp2 expression. Total RNAs and proteins were isolated from kidneys of uninduced Aqp2+/+ and Aqp2ECE/+ kidneys with free water access (n = 3 mice/genotype, A–C) and under water deprivation for 24 h (n = 4 mice/genotype, D–F). Each RNA sample was analyzed by real-time RT-qPCR in triplicates, using GAPDH as internal control. For Western blotting analyses, rabbit Aqp2 (sc-28629) was used. The 35- to 50-kDa band corresponding to glycosylated Aqp2 was quantified, normalized to β-actin, and presented in C and F, respectively. In all case, relative Aqp2 mRNA and protein levels in WT were set to 1. *P < 0.05 vs. WT.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Aqp2ECE possesses absolute no leaky activity. A–C: immunofluorescence staining for Aqp2 (green) and red fluorescent protein (RFP; red) showing no RFP expression in whole kidney of an oil-injected adult ECE/+RFP/RFP mouse. Cells indicated by arrowheads were magnified in the insets. Scale bar, 50 μm, 8.3 μm for inset.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Activity Assay, Immunofluorescence, Staining, Expressing, Injection

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: There is no indication of extrarenal Aqp2 expression and ECE-mediated recombination. A–I: immunofluorescence staining for Aqp2 (green) and RFP (red) showing lack of their expression in all organs except kidney, as indicated in adult ECE/+RFP/RFP mice treated with a single dose of tamoxifen (2 mg) for 24 h. Cells indicated by arrowheads are magnified in the insets. DAPI staining (blue) for nuclei was added. Scale bar, 50 μm, 8.3 μm for inset for each panel.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Immunofluorescence, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Aqp2ECE has complete fidelity in recapitulating the cell-specific expression pattern of Aqp2. A–C: immunofluorescence staining for Aqp2 (green) and RFP (red) showing all RFP+ cells were also Aqp2+ in whole kidney of adult ECE/+RFP/RFP mice treated with a single dose of tamoxifen (2 mg) for 24 h. Cells indicated by arrowheads are magnified in the insets. Scale bar, 50 μm, 8.3 μm for inset.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Immunofluorescence, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Aqp2ECE is inactive in intercalated cells marked by V-ATPase subunits B1 and B2 (B1B2). A–C: immunofluorescence staining for Aqp2 (blue) and B1B2 (green) to mark intercalated cells and RFP (red) showing no RFP coexpression with B1B2 in whole kidney of adult ECE/+RFP/RFP mice treated with a single dose of tamoxifen (2 mg) for 24 h. Cells indicated by arrowheads are magnified in the insets. Scale bar, 50 μm, 8.3 μm for inset.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Immunofluorescence, Staining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Highly tamoxifen-inducible principal cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

doi: 10.1152/ajprenal.00436.2017

Figure Lengend Snippet: Aqp2ECE is inactive in intercalated cells marked by carbonic anhydrase II (CAII). A–C: immunofluorescence staining for Aqp2 (blue) and CAII (green) to mark intercalated cells and RFP (red) showing no RFP coexpression with CAII in whole kidney of adult ECE/+RFP/RFP mice treated with a single dose of tamoxifen (2 mg) for 24 h. Cells indicated by arrowheads are magnified in the insets. Scale bar, 50 μm, 8.3 μm for inset.

Article Snippet: Two mouse antibodies specific for carbonic anhydrase II (CAII, sc-48351) and V-ATPase B1 B2 (sc-55544), one rabbit Aqp2 antibody (sc-28629), and two goat antibodies against Aqp2 (sc-9882) and Pendrin (sc-16894) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Immunofluorescence, Staining

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: Journal of Clinical Investigation

Article Title: Krüppel-like factor 4 regulates macrophage polarization

doi: 10.1172/jci45444

Figure Lengend Snippet: Figure 1 Klf4 expression is augmented by M2 and inhibited by M1 stimuli in macrophages. (A and B) KLF mRNA levels in (A) mouse PMs and (B) BMDMs after 16 hours of stimulation with LPS (50 ng/ml) or mouse IL-4 (5 ng/ml). Gene expression levels were assessed by qPCR and normalized to those in untreated cells (dashed line). Klf1, Klf15, and Klf17 were not detectable in PMs or BMDMs. Klf5 and Klf14 were not detectable in BMDMs. n = 5. (C and D) KLF4 protein levels after LPS and IL-4 treatment. n = 3. (E) KLF4 mRNA level in M-CSF–differentiated primary human macrophages (from 3 donors) stimulated with LPS (50 ng/ml) or human IL-4 (10 ng/ml) for 16 hours. (F) IL-4–mediated induction of the Klf4 gene was diminished in Stat6-null PMs and BMDMs. n = 3. C57BL/6J mice were used as control (WT). *P < 0.05, Student’s t test.

Article Snippet: Other antibodies were iNOS (SC-8310), Cox-2 (SC-7951), PPARγ (SC7273), and HRP-conjugated anti-goat IgG (SC-2020) from Santa Cruz Biotechnology Inc.; Stat6 (catalog 9362) and phospho–Tyr641-Stat6 (catalog 9361) from Cell Signaling Technology; goat anti-KLF4 (AF3158) from R&D Systems; mouse anti–β-actin (A1978) from Sigma-Aldrich; HRP-conjugated anti-rabbit IgG (NA934V) and anti-mouse-IgG (NA931V) from GE Healthcare; and HRP-conjugated anti-chicken IgY (SA1-300) from Affinity BioReagents.

Techniques: Expressing, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: Krüppel-like factor 4 regulates macrophage polarization

doi: 10.1172/jci45444

Figure Lengend Snippet: Figure 2 KLF4 is essential for IL-4–mediated macrophage M2 polarization. (A) Impairment of M2 marker gene expression in KLF4-deficient macrophages. PMs isolated from Mye-WT and Mye-KO mice were treated with IL-4 for 16 hours. n = 3 in each group. (B) KLF4 overexpression enhances M2 gene expression. RAW264.7 cells were infected with either Ad-GFP (Ad-EV) or Ad-KLF4 for 24 hours prior to treatment with IL-4 for 16 hours. n = 3 in each group. (C) Representative Western blot showing IL-4–mediated protein induction of Arg-1, Retnla, Chi3l3, and PPARγ in Mye-WT and Mye-KO macrophages. (D) Quantification of Western blot data by densitometry. For Arg-1, protein levels were normalized to Mye-WT control group. For Retnla, Chi3l3 and PPARγ, only IL-4–induced protein levels were calculated and normalized to the IL-4–treated Mye-WT group, due to extremely low levels of expression at baseline. Data were calculated from 3–5 independent blots. (E) Synergistic activation of the mouse Arg1 promoter by KLF4 and Stat6 as assayed by transient transfection. WT, ~4 kb WT mouse Arg1 promoter; ΔKLF, Arg1 promoter with both KLF- binding sites mutated; ΔStat6: Arg1 promoter with Stat6-binding site mutated. Transient transfection experiments were performed in RAW264.7 cells. n = 3. (F) KLF4 binding to Arg1 promoter detected by ChIP assay in WT and Stat6-null BMDMs with or without IL-4 treatment (4 hours). *P < 0.05, **P < 0.01, Student’s t test with Bonferroni correction.

Article Snippet: Other antibodies were iNOS (SC-8310), Cox-2 (SC-7951), PPARγ (SC7273), and HRP-conjugated anti-goat IgG (SC-2020) from Santa Cruz Biotechnology Inc.; Stat6 (catalog 9362) and phospho–Tyr641-Stat6 (catalog 9361) from Cell Signaling Technology; goat anti-KLF4 (AF3158) from R&D Systems; mouse anti–β-actin (A1978) from Sigma-Aldrich; HRP-conjugated anti-rabbit IgG (NA934V) and anti-mouse-IgG (NA931V) from GE Healthcare; and HRP-conjugated anti-chicken IgY (SA1-300) from Affinity BioReagents.

Techniques: Marker, Gene Expression, Isolation, Over Expression, Infection, Western Blot, Control, Expressing, Activation Assay, Transfection, Binding Assay