Anti irf9

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-IRF9 is a laboratory reagent that can be used to detect and analyze the expression of the IRF9 protein in biological samples. IRF9 is a transcription factor that plays a crucial role in the regulation of interferon-stimulated genes and the cellular response to viral infections. The anti-IRF9 reagent can be used in various research applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of IRF9 in different cell types and experimental conditions.

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5 protocols using anti irf9

Western Blot Analysis of Signaling Proteins

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Anti-pSTAT1, Anti-p-MAPKAPK-2, Anti-p-ERK, Anti-MAPKAPK-2, and anti-IRF9 for Western blotting were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Calreticulin antibody was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Anti-GAPDH antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Western blotting was done as described previously [70 (link)]. Cells were lysed in TN1 buffer (50 mM Tris (pH 8.0), 10 mM EDTA, 10 mM Na₄P₂O₇, 10 mM NaF, 1% Triton X-100, 125 mM NaCl, 10 mM Na₃VO₄, and 10 μg/ml each aprotinin and leupeptin). Protein lysates were boiled in Laemmli sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with the antibody of interest, and then developed by Pierce ECL 2 Western blotting substrate (Thermo Scientific, Rockford, IL, USA) or SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific). Densitometry was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and ratios between the indicated probes and their respective anti-actin reprobes were calculated.

Fatehchand K., Mehta P., Colvin C.B., Buteyn N.J., Santhanam R., Merchand-Reyes G., Inshaar H., Shen B., Mo X., Mundy-Bosse B., Tridandapani S, & Butchar J.P. (2021). Activation of plasmacytoid dendritic cells promotes AML-cell fratricide. Oncotarget, 12(9), 878-890.

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Protein Expression Analysis in PCa Cells

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Total protein was extracted from the PCa cell pellets with RIPA Lysis Buffer (Beyotime biotechnology, Shanghai, China). Protein samples were loaded into 10% SDS–PAGE and subjected to electrophoretic analysis and subsequent blocking. Membranes were incubated with the primary antibodies (overnight at 4 °C) and the relevant secondary antibodies (1 h at room temperature). The antibodies were used in this study as follows: anti-AR (Santa Cruz biotechnology, Inc., USA, 1:1000), anti-FAM64A (Thermo Fisher Scientific, Inc., USA, 1:1000), anti-cyclin B1 (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-β-tubulin (Abcam, UK, 1:5000), anti-stat1 (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-phospho-stat1 (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-stat2 (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-phospho-stat2 (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-IRF-9 (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-rabbit secondary antibody (Cell Signaling Technology, Inc., MA, USA, 1:1000), anti-mouse secondary antibody (Cell Signaling Technology, Inc., MA, USA, 1:1000).

Zhou Y., Ou L., Xu J., Yuan H., Luo J., Shi B., Li X., Yang S, & Wang Y. (2021). FAM64A is an androgen receptor-regulated feedback tumor promoter in prostate cancer. Cell Death & Disease, 12(7), 668.

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Immunoblotting Analysis of Protein Expression

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Immunoblotting was performed according to standard procedures. Total proteins were extracted from treated cells lysed with ice-cold radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors. The amounts of proteins were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies used included anti-PTBP2, anti-PTBP1 antibody, anti-IRF9, anti-STAT1, anti-vinculin (#13901, Cell Signaling Technology, Beverly, USA), anti-STAT2 (#16674-1-AP, Proteintech, Chicago, IL, USA), anti-β-actin (#4790, Cell Signaling Technology, Beverly, USA), and anti-histone H3 (#4499, Cell Signaling Technology, Beverly, USA). All the bands were visualized by enhanced chemiluminescence, replicated at least 3 times, and quantified by ImageJ analysis software.

Tang J., He J., Guo H., Lin H., Li M., Yang T., Wang H.Y., Li D., Liu J., Li L., Xia H., Zhuo Z, & Miao L. (2023). PTBP2-Mediated Alternative Splicing of IRF9 Controls Tumor-Associated Monocyte/Macrophage Chemotaxis and Repolarization in Neuroblastoma Progression. Research, 6, 0033.

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Protein Expression Analysis by Western Blot

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Protein extracts were prepared using RIPA buffer (Sigma: 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris, pH8.0) completed with Protease Inhibitor (PI) Cocktail tablet (Sigma). Protein concentration was determined with the Pierce BCA protein assay kit (Thermo Scientific). Protein extracts (20-40 µg) were fractionated by SDS polyacrylamide gel electrophoresis on 4–12% Bis–Tris precast polyacrylamide gels (Invitrogen, Paisley, UK) and transferred to a nitrocellulose membrane (Invitrogen). Bands were visualized by chemiluminescence (ECL Plus; GE Healthcare, Hatfield, UK). Protein quantification was performed using ImageJ and statistical significance was assessed using GraphPad Prism 8. Anti-IRF2 (Abcam, cat#ab124744, 1:500 dilution), anti-IRF9 (Cell Signaling, cat#76684, 1:500 dilution), anti-YY1 (Cell Signaling, cat#2185, 1:500 dilution), anti-SNAI2 (Abcam, cat#ab27568, 1:200 dilution), and anti-p16 (CDKN2A, Abcam, cat#ab54210, 1:1000 dilution) antibodies were used.

Mercado N., Schutzius G., Kolter C., Estoppey D., Bergling S., Roma G., Gubser Keller C., Nigsch F., Salathe A., Terranova R., Reece-Hoyes J., Alford J., Russ C., Knehr J., Hoepfner D., Aebi A., Ruffner H., Beck T.C., Jagannathan S., Olson C.M., Sheppard H.E., Elsarrag S.Z., Bouwmeester T., Frederiksen M., Lohmann F., Lin C.Y, & Kirkland S. (2019). IRF2 is a master regulator of human keratinocyte stem cell fate. Nature Communications, 10, 4676.

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Keratinocyte Protein Extraction and Analysis

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Keratome biopsies were homogenized in a cell lysis buffer (20 mM Tris-base (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodidum pyrophosphate, 1 mM β-glycerolphosphate, 1mM Na₃VO₄, 1 μg/ml leupeptin, and 1 mM PMSF) as previously described [15 (link)]. The samples were then centrifuged at 10,000 x g for 10 minutes at 4°C, after which the supernatant constituted the cell lysate. The protein extracts from cultured human keratinocytes were isolated as previously described [16 (link)].
Equal protein amounts were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with anti-phospho-STAT2(Tyr690), anti-STAT2, anti-STAT1 (cat. no. 4441, 4594, and 9172, respectively; Cell Signaling Technology, Danvers, MA), anti-IRF9 (cat. no. AF5629, R&D Systems, Oxon, UK), or anti-β-actin (cat. no. A-1978; Sigma-Aldrich, St. Louis, MO). The antibodies were detected with anti-rabbit IgG-HRP (cat. no. 7074; Cell Signaling Technology, Danvers, MA) or with anti-sheep IgG-HRP (cat. no. P0163; Dako, Glostrup, Denmark) in a standard ECL reaction (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. Densitometric analysis of the band and background intensities was conducted using Kodak 1D Image analysis software. Results were normalized to β-actin levels.

Johansen C., Rittig A.H., Mose M., Bertelsen T., Weimar I., Nielsen J., Andersen T., Rasmussen T.K., Deleuran B, & Iversen L. (2017). STAT2 is involved in the pathogenesis of psoriasis by promoting CXCL11 and CCL5 production by keratinocytes. PLoS ONE, 12(5), e0176994.

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