Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Virus infection induces delayed STAT1 Ser-708 phosphorylation. A, WT or IKKϵ−/− MEFs were mock-stimulated or stimulated with 100IU/ml IFN-β. Protein lysate was collected 16 h post-IFN stimulation and immunoblotted using p-STAT1 Ser-708, p-STAT1 Ser-727, p-STAT1 Tyr-701, and total STAT1 antibodies. B and C, Sendai and WNV virus infections induce STAT1 Ser-708 phosphorylation. HEK293 cells were infected with 100 HA units/ml SenV (B) or West Nile virus strain Madagascar (WNV-MAD) (C) at an m.o.i. of 1. At the indicated times following infection, protein lysates were collected and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, p-IRF-3, total IRF-3, IFIT1, and SenV or WNV. Tubulin and GAPDH were used as loading controls.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Type I, type II, and type III IFNs induce STAT1 Ser-708 phosphorylation. 2fTGH cells were mock-treated or treated with 100 IU/ml IFN-β (A) or 50 ng/ml IFN-γ (B). C, PH5CH8 cells were mock-treated (lane 1), treated with 100 ng/ml IFN-λ1 (lanes 2-6), or 100 IU/ml IFN-β (lanes 7-9). Protein lysate was collected at respective time points following IFN treatment and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: Signaling through IFNAR is required for STAT1 Ser-708 phosphorylation following type I IFN treatment or virus infection. WT, IRF-3−/−, or IFNAR−/− (A) and parental 2fTGH cells or their derivative U5A cells (which lack IFNAR) (B) were infected with WNV-MAD at an m.o.i. of 1. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, and WNV. C and D, the same cells were also mock-stimulated or stimulated with 100 IU/ml IFN-β for 6 or 16 h. Protein lysates were collected at the indicated time points and immunoblotted for p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, p-IRF-3, total IRF-3, IFIT2, IFIT3, and IFIT1. Asterisk, nonspecific band.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: STAT1 Ser-708 phosphorylation requires de novo protein synthesis, STAT1 tyrosine dephosphorylation, and nuclear export. A, 2fTGH cells were mock-treated (-CHX, lanes 1-5) or treated with CHX (+CHX, lanes 6-11) to block protein synthesis. At 30 min (lanes 6-10) or 16 h (lane 11) following CHX treatment, cells were mock-stimulated (M) or stimulated with IFN-β. Cells were harvested at 10 min as well as 1, 6, and 16 h post-IFN stimulation and immunoblotted to detect p-STAT1 Ser-708, p-STAT1 Tyr-701, total STAT1, ISG15, and IFIT1. B, 2fTGH cells were not treated (NT, lanes 1-3), pretreated with 100 nm LMB (lanes 4-6), or 50 mm pervanadate (Van, lanes 7-9) 1 h before mock stimulation or stimulation with 100 IU/ml IFN-β. Cells were harvested at 1 and 16 h post-stimulation. Immunoblot analysis was performed using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, ADAR1, and IFIT1 antibodies.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: De-Phosphorylation Assay, Blocking Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: IKKϵ mediates IFIT2 expression and protection against WNV pathogenesis in vivo. WT Bl6 and IKKϵ−/− mice were mock-infected (PBS only) or infected with 103 pfu of WNV-MAD subcutaneously through footpad injection. A, mice were monitored and scored daily for clinical symptoms over 17 days. Clinical scores from four representative mice per group were graphed. B, spleens from WT or IKKϵ−/− mice, mock-infected or infected with WNV-MAD, were collected at days 4, 6, and 12 post-infection. Protein lysates were extracted by homogenizing spleens with radioimmune precipitation assay buffer and immunoblotted using p-STAT1 Ser-708, p-STAT1 Tyr-701, p-STAT1 Ser-727, total STAT1, IFIT2, IFIT1, WNV, and IKKϵ antibodies. The immunoblot analysis panel is a representative from four mice per infection group.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Expressing, In Vivo, Infection, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Inhibitor of ?B Kinase ? (IKK?), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection *

doi: 10.1074/jbc.M111.285205

Figure Lengend Snippet: A model illustrating that early and late ISGs induction is regulated by multiple STAT1 posttranslational modifications. 1, the canonical JAK-STAT signaling is activated following type I IFN binding to its receptor, which results in STAT1 Tyr-701 phosphorylation, ISGF3 formation, and its nuclear translocation. ISGF3 binding to the ISRE element induces transcription of ISGs. 2, chromatin-bound STAT1 can be phosphorylated by MAPK at residue Ser-727, which induces its sumoylation. 3 and 4, nuclear STAT1 is also acetylated by histone acetyltransferase (HAT) CREB-binding protein (CBP), resulting in recruitment of TCP1, which catalyzes STAT1 tyrosine dephosphorylation. Sumoylated-acetylated STAT1 cycles back to the cytoplasm, and both modifications render STAT1 unable to be further tyrosine-phosphorylated. 5 and 6, type I IFN signaling and unknown IFN-stimulated factor(s) activate IKKϵ phosphorylation of STAT1 Ser-708. 7, STAT1 molecules phosphorylated at Ser-708 can enter the nucleus and induce expression of a specific ISG subset. pY, tyrosine phosphorylation; pS, serine phosphorylation; Ac, acetylation; Su, sumoylation.

Article Snippet: Affinity-purified rabbit polyclonal anti-STAT1 Ser-708 antibody was generated by repeat-immunization with the STAT1 Ser-708 phospho-specific peptide (YIKTELI{pS}VSEVHP, amino acids 701–714) (GenScript).

Techniques: Binding Assay, Translocation Assay, De-Phosphorylation Assay, Expressing

Journal: medRxiv

Article Title: Neonatal-onset autoinflammation and immunodeficiency caused by heterozygous missense mutation of the proteasome subunit β-type 9

doi: 10.1101/2021.02.01.21250077

Figure Lengend Snippet: Panels A and B show the proteasome analysis of unstimulated or IFN-γ-stimulated skin fibroblasts from a healthy control and patient 2. Cell extracts were prepared and fractionated by glycerol gradient centrifugation. Panel A shows immunoblot analysis of each fraction using Abs against the indicated proteins. Panel B shows chymotrypsin-like activity of each fraction, which was measured by using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.0025% SDS. Panel C shows the STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts of control subjects and Patient 2. The expression levels of IFN-γ induced p-STAT1 were enhanced in patient 2 compared to control subjects. Panel D shows the skin biopsy sample of patient 2 stained with anti-ubiquitin Ab. A scale bar represents 100 μm.

Article Snippet: Six μm sections of biopsy samples from the patients’ skin lesion were stained with rabbit polyclonal anti-p-STAT1 Abs (GeneTex) and anti-ubiquitin Abs (DAKO).

Techniques: Control, Gradient Centrifugation, Western Blot, Activity Assay, Phospho-proteomics, Transformation Assay, Expressing, Staining, Ubiquitin Proteomics

Journal: Journal of Inflammation Research

Article Title: Huiyang Shengji Unguent Promotes Lymphangiogenesis and Wound Healing in Diabetic Chronic Wounds: Combined Insights from Proteomics and in vivo and in vitro Analyses

doi: 10.2147/JIR.S534105

Figure Lengend Snippet: HYSJ inhibits activation of the pyroptosis pathway in HLECs in a high glucose inflammation model. ( A ) Western blot analysis of upstream proteins involved in pyroptosis, including TLR2, Myd88, and Stat1, in HLECs under the indicated conditions. ( B – D ) Relative expression level analysis of TLR2, Myd88, and Stat1 proteins. ( E ) Pyroptosis pathway proteins NLRP3, caspase-1, gasdermin D, and N-gasdermin D expression. ( F – I ) Relative expression of pyroptosis pathway proteins NLRP3, caspase-1, gasdermin D, and Ngasdermin D. ( J ) Immunofluorescence detection of N-gasdermin D expression in HLECs under the indicated intervention conditions. ( K ) Fluorescence intensity analysis of Ngasdermin D in each group. ( L ) LDH levels in supernatants of HLECs under the indicated intervention conditions. Each group n=3. *P < 0.05, **P < 0.01, ***P<0.001, ****P < 0.0001 compared to model group.

Article Snippet: The membrane was incubated with the following primary antibodies as required for 10 hours at 4 °C:recombinant anti-NLRP3 antibody (1:1000; Cat. No. 4691S, Abcam, Cambridge, UK), caspase-1 (E9R2D) rabbit mAb (1:1000; Cat. No. 83383; Cell Signaling Technology, Boston, USA), gasdermin D (E9S1X) rabbit mAb (1:500; Cat. No. HA721144; HUAbio, Hangzhou, China), recombinant anti-LYVE1 antibody RM1067 (1:1000; Cat. No. ab314241; Abcam), PROX1 recombinant rabbit monoclonal antibody (1:500; Cat. No.HA722318; HUAbio), Flt-4 rabbit polyclonal antibody (1:1000; Cat. No. ER65750; HUAbio), VEGFC rabbit polyclonal antibody (1:1000; Cat. No. 22601–1; Proteintech, Wuhan, China), STAT1 recombinant rabbit monoclonal antibody (1:1000; Cat. No. ET1612; HUAbio), MyD88 rabbit pAb (1:1000; Cat. No. A0980; Abclonal, Wuhan, China), TLR2 recombinant rabbit monoclonal antibody (1:800; Cat. No. ET1705; HUAbio), or β-actin antibody (1:8000; Cat. No. #52901; Signalway Antibody, Maryland, USA).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Dietary Lauric Acid Suppresses Inflammation, Cholestasis, Hepatocyte Injury, and Senescence in 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-induced Inflammatory Cholangiopathy

doi: 10.1016/j.jcmgh.2026.101731

Figure Lengend Snippet: Effect of lauric acid on expression of inflammation, senescence, and oxidative stress genes and biomarkers in hepatocytes isolated from experimental mouse groups. qRT-PCR-mRNA analysis in hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( A ) Genes involved in inflammation and oxidative stress. ( B ) Senescence genes. ( C ) Mitochondrial fatty acid oxidation and oxidative stress genes. ( D ) β-gal staining ( yellow arrow ) as a senescence marker in overnight cultured primary hepatocytes isolated from chow, DDC, LA, and DDC/ LA mice. Bar graph showing the number of senescent (β-gal + ) cells per microscopic field in liver sections from chow, DDC, DDC/LA, and LA groups. ( E ) Immunoblotting and quantification of β-gal in fresh hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( F ) Serum hydrogen peroxide levels and CCL2 concentrations (ELISA), and lipid peroxides, nitrite, and malondialdehyde levels from isolated hepatocytes. ( G–H ) Co-culture of human cholangiocytes H69 ( upper wells ) and Huh7 cells was conducted. H69 cells were incubated with and without DDC overnight in the presence or absence of DLPC. ( G ) mRNA expression by qPCR was measured in Huh7 cells ( bottom wells ) for CCL2, CDKN1A, NR0B2, and ABCB11 and ( H ) in H69 cells for CCL2, CDKN1A, KRT19, CTGF, and TNF . ( I ) Primary mouse hepatocytes were incubated with CCL2 in the presence or absence of DLPC overnight, and mRNA expression by qPCR was analyzed for senescence genes Cdkn1a and Cdkn1b . ( J ) β-gal staining ( yellow arrow = senescent cells) of cultured Huh7 cells incubated with CCL2 overnight in the presence or absence of DLPC. Bar graph showing the number of senescent (β-gal + ) Huh7 cells after treatment with CCL2 alone or in combination with DLPC. CCL2 treatment markedly increased cellular senescence, which was inhibited by DLPC. ( K ) Confocal fluorescence microscopy of primary cultured hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P16 ( red ), and nuclear DAPI ( blue ) from showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. ( L ) Confocal fluorescence microscopy of primary mouse hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P21/WAF/CIP ( red ) and nuclear DAPI ( blue ) also showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. For ( G–I ), data points represent replicates in 3 independent experiments. For ( D and J ), photomicrographs are shown that are representative of 3 separate experiments. For ( K and L ), immunofluorescent images are shown that are representative of 3 separate experiments. ( M ) Western analysis of pSTAT1 protein, total STAT1, and actin expression in hepatocytes isolated from the mouse groups, including quantification of integrated density values (IDVs). ( N ) ChIP assay of hepatocytes isolated from mouse groups for STAT1 binding to the promoter region of Cdkn1b using a STAT1-specific antibody. ( O ) Western analysis of pSTAT1 protein expression in cultured primary mouse hepatocytes exposed to CCL2 overnight in the presence or absence of DLPC, including quantification of IDVs of immunoblots. Statistical analysis was performed by 1-way ANOVA with Tukey’s correction for multiple comparisons. a P < .05 vs all other groups; b P < .05 vs DDC; b P < .05 vs CCL2.

Article Snippet: Phospho-STAT1 , 9167 , 1:1000 , Cell Signaling.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Staining, Marker, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: ( A ) Spleen leukocytes were stimulated with PHA for 12 h. Relative mRNA levels of T-bet, STAT1 and STAT4 were examined by qPCR, n = 4. ( B ) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with PHA. ( C ) Transcriptional levels of IFN-γ in HEK 293T cells that transfected with tilapia T-bet, STAT1 and STAT4, n = 6. ( D ) HEK 293T cells were co-transfected with tilapia T-bet, STAT1 or STAT4, and pGL3-IFN-γ promoter. The LUC activities were assessed at 48 h post-transfection, n = 4. ( E-G ) Spleen leukocytes from tilapia that i . p . injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with CD3ε mAb for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ ( E, F , n = 4), and the percentage of CD3 + CD4-1 + IFN-γ + T cells ( G ) were examined. ( H-L ) Tilapia i . p . injected with T-bet-specific, STAT1-specific or control siRNA were infected with E . piscicida . Tilapia was i . p . injected with BFA 6 h before sacrifice, and spleen leukocytes were harvest for assay. Western blot assay showed the expression of IFN-γ at 48 h post infection ( H ). Flow cytometry and scatter plot figures showed the percentage and absolute numbers of CD3 + CD4-1 + T cells (I, K) and CD3 + CD4-1 + IFN-γ + T cells (J, L) on 5 DPI, n = 4. ( M-O ) Tilapia individuals that infected with E . piscicida were injected with STAT1 inhibitor Fludarabine or PBS, and animals were i . p . injected with BFA 6 hours before sacrifice. ( M ) Western blot assay showed the protein levels of IFN-γ in spleen leukocytes on indicated days. ( N ) Flow cytometry showed the percentage of IFN-γ in spleen CD4-1 + T cells on day 7 post-infection. ( O ) Kaplan-Meyer survival plot showed the survival percentage of tilapia, n = 25. These experiments were repeated for at least two independent times. *: p <0.05, **: p <0.01, ***: p <0.001, determined by a two-tailed Student’s t-test. The accession numbers of selected sequences were listed in .

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: Flow Cytometry, Phospho-proteomics, Transfection, Injection, Control, Infection, Western Blot, Expressing, Two Tailed Test

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: ( A, B ) Spleen leukocytes were stimulated with PHA or CD3ε/CD28 mAbs for 12 h, and mRNA levels of IFNγR1 and IFNγR2 were examined by qPCR, n = 6. ( C ) SDS-PAGE assay showed the recombination of tilapia IFNγR1 and IFNγR2 with GST-tag in E . coli . ( D ) GST pull-down assay showed the interaction of tilapia IFN-γ with IFNγR1 and IFNγR2. ( E ) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with recombinant IFN-γ. ( F, G ) Spleen leukocytes were stimulated with recombinant IFN-γ for 12 h, and mRNA levels of indicated molecules were examined by qPCR, n = 5. ( H ) Tilapia was injected with STAT1 inhibitor for 2 days before spleen leukocytes were stimulated with recombinant IFN-γ for 12 h. The expression levels of T-bet were examined by qPCR, n = 6. ( I-K ) Spleen leukocytes from tilapia that i . p . injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with recombinant IFN-γ for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ ( I, J , n = 5), and the percentage of IFN-γ + cells in gated CD3 + CD4-1 + T cells ( K ) were examined. These experiments were repeated for at least two independent times. *: p <0.05, **: p <0.01, ***: p <0.001, determined by a two-tailed Student’s t-test.

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: SDS Page, Pull Down Assay, Flow Cytometry, Phospho-proteomics, Recombinant, Injection, Expressing, Control, Two Tailed Test

Journal: PLOS Pathogens

Article Title: IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

doi: 10.1371/journal.ppat.1010913

Figure Lengend Snippet: IL-2-mTORC1 signaling coordinates STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in tilapia.

Article Snippet: For p-STAT1 staining, leukocytes were fixed with Foxp3 Fixation/Permeabilization working solution (eBioscience) on ice for 2 h, and then stained with Alexa Fluor 647 conjugated-p-STAT1 (Tyr701) Rabbit mAb (CST) on ice for 30 min. Stained cells were then washed twice with 1× permeabilization buffer.

Techniques: Cell Differentiation

Journal: American journal of physiology. Renal physiology

Article Title: The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

doi: 10.1152/ajprenal.00428.2015

Figure Lengend Snippet: Fig. 1. Polycystin-1 C-terminal tail (PC1- CTT), phosphorylated (p)-JAK2, p-STAT1, and complement factor B (CFB) expression in polycystic kidney disease (PKD). A: p-JAK2, total JAK2, p-STAT1, total STAT1, CFB, and PC1-CTT in control and human autosomal dominant (AD) PKD kidneys were analyzed by Western blotting and then quantified by densitometry. A representative Western blot is shown, and results represent an average of 3 independent experiments. NM, nonmutated. B: p-JAK2, total JAK2, p-STAT1, total STAT1, CFB, and PC1-CTT in / and Cy/ Han: SPRD rat kidneys were analyzed by Western blotting and then quantified by densitometry. Representative Western blot is shown, and results represent an average of 3 independent experiments. WT, wild-type. C: immunohisto- chemistry staining of p-JAK2, p-STAT1, CFB, and PC1-CTT in Han:SPRD rat kidneys is shown. One representative of 3 independent experiments is shown. Bars 40 m. NS, not significant. *P 0.05. **P 0.01. ***P 0.001.

Article Snippet: Total STAT1 (9172), p-JAK2 (3776), and p-STAT1 (7649) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Control, Western Blot, Immunohistochemistry, Staining

Journal: American journal of physiology. Renal physiology

Article Title: The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

doi: 10.1152/ajprenal.00428.2015

Figure Lengend Snippet: Fig. 3. Effect of STAT1 on CFB expression. A: CFB expression in STAT1-overexpressed UCL93 renal epithelial cells 48 h after transfection. Blots were quantified by densitometry, and the average densitometry values from 3 independent experiments is shown. B: CFB expression in renal epithelial cells which were treated with the STAT1 inhibitor fludarabine for 48 h. Blots were quantified by densitometry, and the average densitometry values from 3 independent experiments are shown. One representative Western blot of 3 independent experiments is shown. *P 0.05. **P 0.01. ***P 0.001.

Article Snippet: Total STAT1 (9172), p-JAK2 (3776), and p-STAT1 (7649) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Transfection, Western Blot

Journal: American journal of physiology. Renal physiology

Article Title: The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

doi: 10.1152/ajprenal.00428.2015

Figure Lengend Snippet: Fig. 4. A: representation of CFB promoter with putative STAT binding site (219/210). B: luciferase activity of CFB promoter (CFB-Pr) or mutated CFB promoter (CFB-Pr-Mut) was measured in control or STAT1-transfected UCL93 cells. C: luciferase activity of CFB-Pr, GAS promoter (GAS-Pr), or mutated GAS promoter (GAS-Pr-Mut) was measured in control or STAT1-transfected UCL93 cells. D: a chromatin immunoprecipitation (ChIP) assay was conducted of 293T cells transfected with control or STAT1. Two different regions (700/350, 400/100) were amplified by PCR after ChIP using an anti-STAT1 antibody. One representative of 3 independent experiments is shown. **P 0.01.

Article Snippet: Total STAT1 (9172), p-JAK2 (3776), and p-STAT1 (7649) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Binding Assay, Luciferase, Activity Assay, Control, Transfection, Chromatin Immunoprecipitation

Journal: American journal of physiology. Renal physiology

Article Title: The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

doi: 10.1152/ajprenal.00428.2015

Figure Lengend Snippet: Fig. 5. Effect of PC1-CTT on JAK2/STAT1 activation and CFB expression. A: JAK2/ STAT1 activation and CFB expression in UCL93 renal epithelial cell lines after 48-h PC1-CTT transfection. Blots were quantified by densitometry, and the average densitom- etry values from 3 independent experiments are shown. B: PC1-CTT was transfected in renal epithelial cells and followed by 24-h fludarabine treatment. CFB expression was an- alyzed by Western blotting and then quantified by densitometry. The average densitometry values from 3 independent experiments are shown. One representative Western blot of three independent experiments is shown. *P 0.05. **P 0.01. ***P 0.001.

Article Snippet: Total STAT1 (9172), p-JAK2 (3776), and p-STAT1 (7649) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Expressing, Transfection, Western Blot

Journal: American journal of physiology. Renal physiology

Article Title: The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

doi: 10.1152/ajprenal.00428.2015

Figure Lengend Snippet: Fig. 6. A: luciferase activity of CFB-Pr or CFB-Pr-Mut was measured in control or PC1-CTT-transfected renal epithelial cells. B: cotransfection of renal epithelial cells with DN-STAT1 (DN1-STAT1 or DN2-STAT1) attenuated PC1-CTT-induced CFB-Pr activity. C: luciferase activity of CFB-Pr, GAS-Pr, or GAS-Pr-Mut was measured in control or PC1-CTT-transfected UCL93 cells. One representative of three independent experiments is shown. *P 0.05. **P 0.01.

Article Snippet: Total STAT1 (9172), p-JAK2 (3776), and p-STAT1 (7649) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Luciferase, Activity Assay, Control, Transfection, Cotransfection

Journal: American journal of physiology. Renal physiology

Article Title: The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

doi: 10.1152/ajprenal.00428.2015

Figure Lengend Snippet: Fig. 7. PC1-CTT promotes RAW264.7 macrophages toward M2 phenotype differentiation through STAT1 and CFB. A: bioactive human CFB was added to unconditioned media to treat macrophages. The expression of the macrophage M1 phenotype marker inducible nitric oxide synthase (iNOS) and M2 phenotype marker arginase-1 were examined by Western blotting. Blots were quantified by densitometry, and the average densitometry values from 3 independent experiments are shown. B: conditioned media were prepared from control (Control-CM) or PC1-CTT (PC1-CTT-CM)-transfected renal epithelial cells. Isotype IgG or CFB antibody was added to treat macrophages. C: conditioned media were generated from renal epithelial cells treated with fludarabine and used to treat macrophages for 24 h. D: PC1-CTT was transfected in renal epithelial cells and followed by fludarabine treatment, and conditioned media were collected to treat macrophages. One representative of 3 independent experiments is shown. *P 0.05. **P 0.01. ***P 0.001.

Article Snippet: Total STAT1 (9172), p-JAK2 (3776), and p-STAT1 (7649) antibodies were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Marker, Western Blot, Control, Transfection, Generated