Ham’s F-12 (1×) nutrient mixture (catalogue number 11765-054), RPMI 1640 medium (catalogue number 11875-093), fetal bovine serum (FBS; catalogue number 16000-044), antibiotic/antimycotic solution (containing 10,000 U/ml penicillin, 10 mg/ml streptomycin, and 25 μg/ml Fungizone®), minimum essential medium nonessential amino acids, l -glutamine, and TrypLE (containing trypsin and ethylenediaminetetraacetic acid) were obtained from Life Technologies (Grand Island, NY, USA). Insulin (bovine pancreas), anti-β-actin, and hydrocortisone were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-IFITM1, anti-STAT1, anti-STAT2, anti-BRG1, anti-p-STAT2 (Tyr690), anti-interferon regulatory factor (IRF)-7, anti-IFNα, anti-p21, anti-cyclin D1, and anti-cyclin E antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and rabbit polyclonal and mouse monoclonal secondary antibodies and anti-p-STAT1 (Tyr701) were purchased from Cell Signaling Technology (Danvers, MA, USA). IFITM1 promoter constructs were kindly provided by Dr. Yeon-Su Lee from the Cancer Genomics Branch, National Cancer Center, Goyang-si, South Korea.
Anti p stat1 tyr701
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p-STAT1 (Tyr701) is a laboratory reagent that detects the phosphorylated form of STAT1 at tyrosine 701. It is used to measure the activation of the STAT1 transcription factor in cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti stat1, by Cell Signaling Technology (7 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti survivin, by Cell Signaling Technology (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti bcl xl, by Santa Cruz Biotechnology (2 mentions)
Anti flag, by Cell Signaling Technology (2 mentions)
Anti p21, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Minimum essential medium non essential amino acids, by Thermo Fisher Scientific (1 mentions)
Anti ifitm1, by Santa Cruz Biotechnology (1 mentions)
Anti stat1, by Santa Cruz Biotechnology (1 mentions)
Anti brg1, by Santa Cruz Biotechnology (1 mentions)
Bovine pancreas insulin, by Merck Group (1 mentions)
Anti ifnα, by Santa Cruz Biotechnology (1 mentions)
Anti stat2, by Santa Cruz Biotechnology (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Anti cyclin e, by Santa Cruz Biotechnology (1 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (1 mentions)
12 protocols using anti p stat1 tyr701
1
Characterization of IFITM1 Regulation
2
Comprehensive Neutrophil Phenotyping and Signaling
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Harvested cells were stained with antibodies including: anti-Ly6G (1:200 dilution; BioLegend, no. 127606, 127610, or 127618), anti-CD11b (1:200 dilution; BioLegend, no. 101206), anti-CD11a (1:200 dilution; BioLegend, no. 153103), anti-SIRPα (1:200 dilution; BioLegend, no. 37210), anti-CD62L (1:200 dilution; BioLegend, no. 104428), anti-PD-L1 (1:200 dilution; BioLegend, no. 124312), anti-CXCR2 (1:300 dilution; BioLegend, no. 149604), anti-ICAM1 (1:300 dilution; BioLegend, no. 116121), anti-CD29 antibodies (1:300 dilution; BioLegend, no. 102221). In some analysis mentioned in the results, cells were treated with a fixation kit (BD Biosciences), subsequently stained with anti-STAT1 (1:100 dilution; Cell Signaling, no. 80916S), anti-p-STAT1 (Tyr701) (1:100 dilution; Cell Signaling, no. 8009S), anti-p-Src (Tyr416) (1:20 dilution; ThermoFisher, no. MA5-28055), or anti-Fyn (1:50 dilution; Santa Cruz, no. sc-434 FITC). Surface phenotype, transcription factor and intracellular protein levels of Ly6G+ neutrophils were analyzed using FACSCanto II (BD Biosciences). Neutrophil viability was assessed by staining and flow analysis with the annexin V/PI kit (1:4,000 dilution; Thermo Fisher Scientific, no. P3566) as described previously74 (link).
3
SUMO1 Regulation of STAT1 Phosphorylation
4
Western Blot Analysis of Protein Expression
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The whole proteins from jejunums of mice and FHs74Int cells were extracted with RIPA lysis buffer (Beyotime Biotechnology). BCA protein assay kit (Beyotime Biotechnology) was used to determine the concentration of protein. Proteins were separated with SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany) followed by blocking with 5% skim milk (BD/Difco, Sparks, MD, USA). The various primary antibodies used in the experiments were as follows: anti-GPX4 (1:1000; Protein Tech Group, Wuhan, China), anti-ACSL4 (1:1000; ABclonal, Wuhan, China), anti-pSTAT1 (Tyr701) (1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-STAT1 (1:1000; Protein Tech Group), anti-IRF1 (1:1000; ABclonal), anti-AMPKα (1:1000; Cell Signaling Technology), anti-pAMPKα (Thr172) (1:1000; Cell Signaling Technology) and anti-β-actin (1:1000; Protein Tech Group). After incubation with primary antibodies and washing with TBST, membranes were incubated with IRDye-conjugated secondary antibodies (1:10000; Li-COR Biosciences, Lincoln, NE, USA). Images of immunoreactive bands were captured with Odyssey CLx Infrared Imaging system (Li-COR Biosciences) and quantified with ImageJ software (National Institutes of Health).
5
Western Blot Analysis of p-p65 and p-STAT1
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SDS-loading buffer was mixed with the lung lysates in RIPA buffer and denatured at 95 °C for 10 min. Lysates were separated by 10% SDS–PAGE and transferred onto nitrocellulose membranes. Blots were incubated with anti-p-p65 (Ser468) (1:1,000 dilution), and anti-p-STAT1 (Tyr 701) (1:1,000 dilution) (Cell Signaling) and anti-β-actin-HRP (1:2,000 dilution) (Santa Cruz Biotechnology). Proteins were visualized with the enhanced chemiluminescence substrate ECL (Pierce, Thermo Fisher Scientific) and imaged using the ChemiDoc XRS Biorad Imager and Image Lab Software 5.1. Uncropped images are presented in Supplementary Fig. 1 .
6
Immunoblot Analysis of Renal Cell Proteins
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Renal cortex and HK-2 cells were lysed with RIPA Lysis Buffer containing a cocktail of 1% protease and 1% phosphatase inhibitors (ComWin Biotech, Beijing, China), and then quantified using BCA assay reagent kit (Dingguo, Beijing, China). Lysates were subjected to immunoblot analysis using primary antibody: anti-ACTB (Sangon Biotech; D110001), anti-FoxO1 (Abcam; ab39670), anti-pFoxO1 (Sangon Biotech; D155054), anti-STAT1(CST; 9172), anti-pSTAT1 (Tyr701) (Cell Signaling Technology; 9167), anti-TGFβ1 (Cell Signaling Technology; 3711), anti-Collagen I antibody (Abcam; ab6308), anti-fibronectin (Proteintech, 15613-1-AP), anti-E-Cadherin (Cell Signaling Technology; 3195), anti-alpha-Smooth Muscle Actin (Novus; NB600). Protein bands were detected by enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA). Relative protein expression was quantified using Image J.
7
Western Blot Protein Detection Protocol
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Western blot analysis was performed using standard techniques as previously described [6] (link). The following antibodies were employed: anti-PARP(1∶1000), anti-STAT1 (1∶1000) and anti-p-STAT1(Tyr-701)(1∶1000), anti-FLAG (1∶1000), anti-caspase 3 (1∶1000), anti-survivin (1∶1000), anti- Bcl-2 (1∶1000), anti-p21 (1∶1000) and anti-cyclin D1(1∶1000), all of which were purchased from Cell Signaling (Danvers, MA, USA). Anti-Bcl-xL (1∶1000) and anti-ß-actin (1∶1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Densitometric analysis was performed using the Image J analysis system (Bethesda, WA, USA); the values for the bands were normalized to those of the β-actin bands.
8
STAT1/3 Interaction with p300 in CNTF Signaling
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Dissected spinal cords or cells were lysed and analyzed by Western blot analysis [24] (link). Antibodies used were: rabbit anti-STAT3 (Santa Cruz), rabbit anti-STAT1 (Santa Cruz), rabbit anti-pSTAT3 (Tyr 705) and anti-pSTAT1 (Tyr 701) (Cell Signaling Technology), mouse anti-α tubulin (Sigma), and mouse anti-GFAP (Chemicon). IP experiments were performed as described previously [25] (link). HEK-293T cells were transfected with pCDNA3-myc-p300 and pBOS-flag-Stat1 or pBOS-flag-Stat3. After serum starvation, CNTF (100 ng/ml) were treated. After 0.5 or 1.5 hours, cells were lysed in IP lysis buffer with protease inhibitor cocktail (Calbiochem). Lysate were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma). Immunoprecipitates were analyzed by Western blot analysis using anti-Myc- (Cell Signaling Technology) and anti-FLAG M2-Peroxidase (Sigma) antibodies.
9
Comprehensive Immunoblot Antibody Panel
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The following primary antibodies were used for immunoblot: anti-FTH1 (cat no., 3998S), anti-IRP1(cat no., 20272S), anti-STAT1(cat no., 14994) and anti-pSTAT1(Tyr701) (cat no., 9167) were all purchased from Cell Signaling Technology (Danvers, MA, USA); anti-IRP2 (cat no., NB100-1798), and anti-FPN (cat no., NBP1-21502) were from Novus Biologicals (Centennial, CO, USA); anti-iNOS (cat no., ab178945) were from abcam (Cambridge, MA, USA); anti-Arg1 (cat no., 16001-1-AP) were from Proteintech (Wuhan, China).
10
Western Blot Analysis of STAT Signaling Pathway
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Western blot analysis was performed using standard techniques as previously described [11 (link)]. The following antibodies were employed: anti-STAT1 (1:1000) and anti-p-STAT1 (Tyr-701) (1:1000), anti-FLAG (1:1000), anti-caspase 3 (1:1000), anti-survivin (1:1000), anti- BCL-2 (1:1000) anti-p21 (1:1000) and anti-cyclin D1 (1:1000), all of which were purchased from Cell Signaling (Danvers, MA, USA). Anti-STAT3 (1:1000), anti-p-STAT3 (Tyr-705) (1:1000), anti-BCL-xL (1:1000) and anti-ß-actin (1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Densitometric analysis was performed using the ImageJ analysis system (Bethesda, WA, USA); the values for the STAT1 bands were normalized to those of the β-actin bands.
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