Membrane receptor signaling by various ligands, including interferons and growth hormones such as EGF, induces activation of JAK kinases which then leads to tyrosine phosphorylation of the various Stat transcription factors. Stat1 and Stat2 are induced by IFN-α and form a heterodimer which is part of the ISGF3 transcription factor complex. Although early reports indicate Stat3 activation by EGF and IL-6, it has been shown that Stat3β appears to be activated by both while Stat3α is activated by EGF, but not by IL-6. Highest expression of Stat4 is seen in testis and myeloid cells. IL-12 has been identified as an activator of Stat4. Stat5 has been shown to be activated by Prolactin and by IL-3. Stat6 is involved in IL-4 activated signaling pathways. Mutations in the STAT1 molecule can be gain of function (GOF) or loss of function (LOF). Both of them can cause different phenotypes and symptoms. Recurring common infections are frequent in both GOF and LOF mutations. In humans STAT1 has been particularly under strong purifying selection when populations shifted from hunting and gathering to farming, because this went along with a change in the pathogen spectrum. STAT1 loss of function, therefore STAT1 deficiency can have many variants. There are two main genetic impairments that can cause response to interferons type I and III. First there can be autosomal recessive partial or even complete deficiency of STAT1. That causes intracellular bacterial diseases or viral infections and impaired IFN a, b, g and IL27 responses are diagnosed. In partial form there can also be found high levels of IFNg in blood serum. When tested from whole blood, monocytes do not respond to BCG and IFNg doses with IL-12 production. In complete recessive form there is a very low response to anti-viral and antimycotical medication. Second, partial STAT1 deficiency can also be an autosomal dominant mutation; phenotypically causing impaired IFNg responses and causing patients to suffer with selective intracellular bacterial diseases (MSMD).
背景文献
1. Shakya R et al. Hypomethylating therapy in an aggressive stroma-rich model of pancreatic carcinoma. Cancer Res 73:885-96 (2013).
2. Syu LJ et al. Transgenic expression of interferon- in mouse stomach leads to inflammation, metaplasia, and dysplasia. Am J Pathol 181:2114-25 (2012).
序列相似性
Belongs to the transcription factor STAT family.
翻译后修饰
Phosphorylated on tyrosine and serine residues in response to a variety of cytokines/growth hormones including IFN-alpha, IFN-gamma, PDGF and EGF. Activated KIT promotes phosphorylation on tyrosine residues and subsequent translocation to the nucleus. Upon EGF stimulation, phosphorylation on Tyr-701 (lacking in beta form) by JAK1, JAK2 or TYK2 promotes dimerization and subsequent translocation to the nucleus. Growth hormone (GH) activates STAT1 signaling only via JAK2. Tyrosine phosphorylated in response to constitutively activated FGFR1, FGFR2, FGFR3 and FGFR4. Phosphorylation on Ser-727 by several kinases including MAPK14, ERK1/2 and CAMKII on IFN-gamma stimulation, regulates STAT1 transcriptional activity. Phosphorylation on Ser-727 promotes sumoylation though increasing interaction with PIAS. Phosphorylation on Ser-727 by PRKCD induces apoptosis in response to DNA-damaging agents. Phosphorylated on tyrosine residues when PTK2/FAK1 is activated; most likely this is catalyzed by a SRC family kinase. Dephosphorylation on tyrosine residues by PTPN2 negatively regulates interferon-mediated signaling. Upon viral infection or IFN induction, phosphorylation on Ser-708 occurs much later than phosphorylation on Tyr-701 and is required for the binding of ISGF3 on the ISREs of a subset of IFN-stimulated genes IKBKE-dependent. Phosphorylation at Tyr-701 and Ser-708 are mutually exclusive, phosphorylation at Ser-708 requires previous dephosphorylation of Tyr-701.; Sumoylated with SUMO1, SUMO2 and SUMO3. Sumoylation is enhanced by IFN-gamma-induced phosphorylation on Ser-727, and by interaction with PIAS proteins. Enhances the transactivation activity.; ISGylated.; Mono-ADP-ribosylated at Glu-657 and Glu-705 by PARP14; ADP-ribosylation prevents phosphorylation at Tyr-701. However, the role of ADP-ribosylation in the prevention of phosphorylation has been called into question and the lack of phosphorylation may be due to sumoylation of Lys-703.; Monomethylated at Lys-525 by SETD2; monomethylation is necessary for phosphorylation at Tyr-701, translocation into the nucleus and activation of the antiviral defense.
Western blot analysis of STAT1 alpha on different lysates with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/1,000 dilution.
Lane 1: Jurkat cell lysate Lane 2: A431 cell lysate Lane 3: HeLa cell lysate Lane 4: A549 cell lysate Lane 5: SK-Br-3 cell lysate Lane 6: SK-MEL-28 cell lysate Lane 7: HT-29 cell lysate Lane 8: RAW264.7 cell lysate Lane 9: C2C12 cell lysate
Lysates/proteins at 15 µg/Lane.
Predicted band size: 87 kDa Observed band size: 87 kDa
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1606-39) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
☑ Knockdown (KD)
Western blot analysis of STAT1 alpha on different lysates with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/5,000 dilution.
Lane 1: HAP1-parental cell lysate, 10 µg/Lane Lane 2: HAP1-STAT1 alpha KD cell lysate, 10 µg/Lane
Predicted band size: 87 kDa Observed band size: 87 kDa
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1606-39) at 1/5,000 dilution was used in K1803 at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/5,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-39) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded human colon tissue with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/500 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-39) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded human ovary cancer tissue with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/500 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-39) at 1/500 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded human spleen tissue with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/2,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-39) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/2,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1606-39) at 1/2,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunocytochemistry analysis of RAW264.7 cells labeling STAT1 alpha with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of RAW264.7 cells labeling STAT1 alpha.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET1606-39, 1/100) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Immunocytochemistry analysis of HeLa cells labeling STAT1 alpha with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-STAT1 alpha antibody (ET1606-39) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
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