Rabbit anti phospho stat1 tyr701

Manufactured by Cell Signaling Technology

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Rabbit anti-phospho-STAT1 (Tyr701) is a primary antibody that specifically recognizes the tyrosine 701-phosphorylated form of the STAT1 protein. STAT1 is a transcription factor that plays a key role in the JAK-STAT signaling pathway, which is involved in cellular responses to various cytokines and growth factors.

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STAT1 (305 citations) Anti-STAT1 (160 citations) Phospho-STAT1 (94 citations) Anti-phospho-STAT1 (54 citations) Phospho-STAT1 (Tyr701) (35 citations) Rabbit anti-STAT1 (28 citations) Anti-phospho-STAT1 (Tyr701) (12 citations) Rabbit anti-phospho-STAT1 (6 citations) Rabbit anti-phospho-STAT1 (Tyr701) antibody (2 citations) Rabbit anti-phospho-STAT1 (Tyr701) Ab (2 citations)

8 protocols using rabbit anti phospho stat1 tyr701

Immunoblotting of Cell Death Regulators

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The primary antibodies used for immunoblotting were mouse IgG1 anti-caspase-1 (p20) (Adipogen, #AG-20B-0042, 1/2000 dilution), mouse IgG2b anti-NLRP3 (Adipogen, #AG-20B-0014, 1/1000), goat anti-mouse IL-1β (R&D systems, #AF-401-NA, 1/1000), rabbit anti-GAPDH (Sigma-Aldrich, #G9545, 1/20,000), rabbit anti-IRF3 (Cell Signaling, #4302, 1/2000), rabbit anti-phospho-IRF3 (Ser396) (Cell Signaling, #4947, 1/2000), rabbit anti-MAVS (rodent specific) (Cell Signaling, # 4983, 1/1000), rabbit anti-phospho-STAT1 (Tyr701) (Cell Signaling, #9171, 1/1000), rabbit anti-STAT1 (D1K9Y) (Cell Signaling, #14994, 1/3000), rabbit anti-caspase-3 (Cell Signaling, #9662, 1/1000), mouse IgG1 anti-DDX33 (B-4) (Santa Cruz, #sc-390573, 1/1000), rabbit anti-MLKL (phospho S345) (Abcam, #ab196436, 1/1000), rabbit anti-MLKL (D6W1K) (Cell Signaling, #37705, 1/2000), rabbit anti-phospho-DRP1 (Ser616) (D9A1) (Cell Signaling, #4494, 1/1000), mouse IgG1 anti-DRP1 (BD Biosciences, #611113, 1/2000), rabbit anti-RIPK1 (D94C12) (Cell Signaling, #3493, 1/2000), and guinea pig anti-mouse gasdermin D (Adipogen, #AG-25B-0036, 1/1000).

da Costa L.S., Outlioua A., Anginot A., Akarid K, & Arnoult D. (2019). RNA viruses promote activation of the NLRP3 inflammasome through cytopathogenic effect-induced potassium efflux. Cell Death & Disease, 10(5), 346.

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Immunoblotting and Flow Cytometry Antibodies

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The following antibodies were used in immunoblotting: rabbit anti-B7-H3 (#14058), rabbit anti-PD-L1 (#13684), rabbit anti-CD86 (#91882), rabbit anti-CD47 (#63000), rabbit anti-Phospho-Stat1 (Tyr701) (#9167), rabbit anti-Phospho-Stat1 (Ser727) (#8826) and rabbit anti-Phospho-Stat3 (Tyr705) (#9145) were purchased from Cell Signaling Technology (Boston, USA). Rabbit anti-Stat1 (10144-2-AP), rabbit anti-Stat3 (10253-2-AP), rabbit anti-HuR (11910-1-AP), rabbit anti-HMGCR (13533-1-AP) and rabbit anti-TTP (12737-1-AP) were from Proteintech (Chicago, USA). Mouse anti-Galectin-9 (ab153673, abcam, Cambridge, UK), rabbit anti-GAPDH (db106, Diagbio, Hangzhou, China), goat anti-mouse PD-L1 (AF1019, R&D systems, Minnesota, USA). The antibodies for flow cytometry: FITC Rat IgG2b κ isotype control (#400605), FITC Rat IgG2a κ isotype control (#400506), PE Rat IgG2a λ isotype control (#400635), PE anti-mouse PD-L1 (#124307), FITC anti-mouse CD3 (#100204), FITC anti-mouse CD8a (#100705) and PE anti-mouse CD45 (#103106) were form Biolegend (Chicago, USA).

Zhang W., Pan X., Xu Y., Guo H., Zheng M., Chen X., Wu H., Luan F., He Q., Ding L, & Yang B. (2023). Mevalonate improves anti-PD-1/PD-L1 efficacy by stabilizing CD274 mRNA. Acta Pharmaceutica Sinica. B, 13(6), 2585-2600.

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Phosphorylation of STAT Proteins

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Western blotting using indicated anti-mouse target antibodies was performed. For western blotting, brains were homogenized in Pierce RIPA Buffer (Thermo Fisher Scientific, 89900) with the addition of Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100x) (Thermo Fisher Scientific, 78442) and protein concentrations were determined using a colorimetric assay (Bio-Rad, 5000006). 80 micrograms of each sample were subjected to a 12% Mini-PROTEAN^® TGX^™ Precast Protein Gels, 10-well, 50 μl (Bio Rad, 4561043) and proteins were transferred onto nitrocellulose membranes (GenScript, L00224). Membranes were immunoblotted with rabbit anti-phospho-Stat1 (Tyr701) (Cell Signaling, 58D6, 9167), rabbit anti-Stat1 (Cell Signaling, D1K9Y, 14994), rabbit anti-phospho-Stat3 (Tyr705) (Cell Signaling, D3A7, 9145), rabbit anti-Stat3 (Cell Signaling, 79D7, 4904), and rabbit anti-beta-actin (Cell Signaling, 13E5, 4970). Membranes were then probed with Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Thermo Fisher Scientific, G-21234) and proteins were visualized using Clarity Western ECL Substrate (Bio-Rad, 170–5061).

Hargarten J.C., Ssebambulidde K., Anjum S.H., Vaughan M.J., Xu J., Song B., Ganguly A., Park Y.D., Scott T., Hammoud D.A., Olszewski M.A, & Williamson P.R. (2024). JAK/STAT Signaling Predominates in Human and Murine Fungal Post-infectious Inflammatory Response Syndrome. medRxiv.

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Western Blotting and Immunostaining Antibodies

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The following antibodies were used for Western blotting and/or immunostaining: mouse anti-β-actin (BA3R; Invitrogen; RRID AB_10979409), rabbit anti-STAT1 (M-22; Santa Cruz; RRID AB_632434), rabbit anti-ISG15 (H-150; Santa Cruz; RRID AB_2126309), rabbit anti-IFIT1 (Invitrogen catalog no. PA5-27907 for human/nonhuman primate samples; RRID AB_2545383), rabbit anti-IFIT1 (Invitrogen catalog no. PA3-846 for murine samples; RRID AB_1958734), rabbit anti-phospho-STAT1 (Tyr-701) (catalog no. 9171; Cell Signaling Technology, Inc.; used for Western blotting assays; RRID AB_561284), rabbit anti-phospho-STAT1 (Tyr-701) (D4A7; Cell Signaling Technology, Inc.; used for immunofluorescence; RRID AB_10950970), rabbit anti-phospho-STAT1 (Ser-727) (catalog no. 9177; Cell Signaling Technology, Inc.; RRID AB_2197983), and rabbit anti-phospho-IRF3 (Ser-396) (catalog no. 29047; Cell Signaling Technology, Inc.). Antiflavivirus group antigen D1-4G2-4-15 (ATCC HB-112 hybridoma; RRID CVCL_J890) and mouse anti-YFV ascites fluid were used in flavivirus focus-forming assays. Human and murine IFNs α4 and β were prepared in-house as described previously (36 (link)). Lipopolysaccharides (LPSs) from Escherichia coli 0111:B4 were purchased from Sigma. Poly(I:C) was purchased from R&D Systems.

Lane W.C., Dunn M.D., Gardner C.L., Lam L.K., Watson A.M., Hartman A.L., Ryman K.D, & Klimstra W.B. (2018). The Efficacy of the Interferon Alpha/Beta Response versus Arboviruses Is Temperature Dependent. mBio, 9(2), e00535-18.

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RAGE Signaling Pathways in AGT Regulation

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A rabbit anti-RAGE antibody from Novus and a rabbit anti-AGT antibody from IBL were used in this study. A mouse anti-phospho p47phox (ser359) antibody and a rabbit anti-total p47phox antibody were purchased from Thermo Fisher Scientific. To elucidate RAGE signaling pathways involved in AGT regulation in PTC, mouse antiphospho-p42/44 MAPK (ERK 1/2, Thr202/Tyr204), a rabbit anti-p42/44 MAPK, mouse anti-phospho-STAT3 (Tyr705), rabbit anti-STAT3, rabbit anti-phospho-STAT1 (Tyr701), rabbit anti-STAT1, rabbit anti-phospho-p38 MAPK (Thr180/Tyr182), rabbit anti-p38 MAPK, rabbit anti-p65, rabbit anti-phospho-c-Jun (Ser73), rabbit anti-c-Jun, rabbit antiphospho-c-Fos (Ser32), and rabbit anti-c-Fos antibodies were purchased from Cell Signaling Technology and used in this study. In addition, rabbit anti-phospho-p65 (Ser536) antibody from Signalway Antibody was also used. Mouse anti-GAPDH antibody from Abcam was used as an internal control. IRDye-labeled anti-mouse IgG and anti-rabbit IgG antibodies were obtained from Li-Cor as secondary antibodies in Western blot analyses. Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody from Life Technologies was used as a secondary antibody in immunostaining.

Garagliano J.M., Katsurada A., Miyata K., Derbenev A.V., Zsombok A., Navar L.G, & Satou R. (2018). Advanced Glycation End Products Stimulate Angiotensinogen Production in Renal Proximal Tubular Cells. The American journal of the medical sciences, 357(1), 57-66.

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Regulation of STAT1/2 Phosphorylation by IFNL3

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Example 7

HepG2 cells in 6-well plates were untreated or transfected with expression constructs or empty Halo-tag vector, or were treated for 1 hour at 37° C. with 50 ng/ml of recombinant IFNL3. Equal amounts (50 μg/lane) of whole-cell lysates prepared 48 hours post-transfection were used for analysis by Western blotting. Detection was performed with rabbit anti-phospho-Tyr701-STAT1 (Cell Signaling Technology) and rabbit anti-phospho-Tyr689-STAT2 (Millipore) antibodies. The blots were stripped and re-probed with rabbit anti-STAT1 and anti-STAT2 antibodies (Santa Cruz Biotechnology) to measure the levels of total STAT1 and STAT2 proteins. Only treatment with IFNL3 and transient expression with IFNL4 construct induced phosphorylation of STAT1 and STAT2 (FIG. 14).

US10545147B2. Interferon-λ4 (IFNL-4) protein, related nucleic acid molecules, and uses thereof (2020-01-28). The USA, as represented by the Secretary, Dept. of Health and Human Services [US]. Inventors: Liudmila Prokunina [US], Thomas R. O'Brien [US], Brian Muchmore [US], Raymond P. Donnelly [US], Patricia A. Porter-Gill [US].

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Characterizing Immune Signaling Pathways via Antibody Probing

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Anti-NS3 antibodies were described in [34 (link)]. Rabbit anti-phospho-Tyr701-STAT1, rabbit anti-STAT1, rabbit anti-phospho-NF-κB p65 (Ser 536) and rabbit anti-NF-κB p65, diluted 1:1000 for western blot and 1:200 for immunofluorescence, were from Cell Signaling (Beverly, MA, USA). Mouse anti-Actin, diluted 1:10000, was from MP Biomedicals (Santa Ana, CA, USA, cat. #69100). Mouse anti-tubulin-α, diluted 1:1000, was from Biolegend (San Diego, CA, USA, cat. #625901). Alexa-488 and Alexa-555 conjugated secondary antibodies, diluted 1:200, were from ThermoFisher (cat. #A27039 and A28175). HRP-conjugated secondary antibodies, diluted 1:15,000, were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA, cat. #115035003).

Danziger O., Shai B., Sabo Y., Bacharach E, & Ehrlich M. (2016). Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection. Oncotarget, 7(32), 52115-52134.

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Regulation of STAT1/2 Phosphorylation by IFNL3

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Example 7

US10962539B2. Interferon-λ4 (IFNL-4) protein, related nucleic acid molecules, and uses thereof (2021-03-30). The USA, as represented by the Secretary, Dept. of Health and Human Services [US]. Inventors: Liudmila Prokunina [US], Thomas R. O'Brien [US], Brian Muchmore [US], Raymond P. Donnelly [US], Patricia A. Porter-Gill [US].

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