Fixed retinal tissues were sectioned at 10-µm thickness through the optic disk. Sections were blocked with 5% BSA and 1% Triton X-100 in PBS for 1 hour. The slides were then incubated with SYVN1 antibody (1:200; Novus Biologicals), STAT3 antibody (1:500; Cell Signaling Technology), and CD31 antibody (1:100; Servicebio) and then individually incubated with secondary antibody (Alexa 488/594; Cell Signaling Technology; and Donkey anti-goat, 1:100; Servicebio) incubation for 1 hour. Slide nuclei were stained with 4′,6-diamidino-2-phenylindole DAPI (Invitrogen). After each incubation, the cells were washed three times. The fluorescence of the retinas was imaged using a confocal microscope.
Stat3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
The STAT3 antibody is a primary antibody that specifically binds to the STAT3 (Signal Transducer and Activator of Transcription 3) protein. STAT3 is a transcription factor that plays a crucial role in cellular processes such as cell growth, differentiation, and survival. This antibody can be used for various applications, including Western blotting, immunohistochemistry, and immunofluorescence, to detect and analyze the expression and localization of STAT3 in biological samples.
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Lab products found in correlation
P stat3 antibody, by Cell Signaling Technology (5 mentions)
Gapdh antibody, by Cell Signaling Technology (3 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Cryptotanshinone, by Selleck Chemicals (2 mentions)
Cisplatin, by Teva Pharmaceuticals (2 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Agilent Technologies (2 mentions)
Rotenone antimycin a, by Agilent Technologies (2 mentions)
Sta21, by Selleck Chemicals (2 mentions)
Stattic, by Selleck Chemicals (2 mentions)
Antibodies to histone h2b ab52484 and gapdh ab9485, by Abcam (2 mentions)
Antibody to vimentin sc66002, by Santa Cruz Biotechnology (2 mentions)
Xf dmem base medium, by Agilent Technologies (2 mentions)
C18 synergi 4 μm fusion rp 80 lc column, by Phenomenex (2 mentions)
Waters symmetry c18 column, by Waters Corporation (2 mentions)
Wp1066, by Selleck Chemicals (2 mentions)
N ethylmaleimide, by Merck Group (2 mentions)
Draq5, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
37 protocols using stat3 antibody
1
Immunofluorescent Analysis of Retinal Tissues
2
Silencing STAT3 in SW480 Cells
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The human colorectal carcinoma cell line SW480 was cultured in Dulbecco's minimum essential medium (DMEM) containing 10% fetal bovine serum (FBS, GIBCO). All cells were incubated at 37°C in a humidified chamber supplemented with 5% CO2. siRNAs against STAT3 (STAT3-siRNA) and scrambled siRNA-oligonucleotides (siRNA-control) were purchased from Ambion (Austin, TX, USA). STAT3 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).
3
Western Blot Analysis of Protein Targets
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Western blot analysis was performed as described previously [61 (link)]. The primary antibodies used were as follows: ENO1 antibody (mouse; #MAB11222; Abnova, Taipei, Taiwan), STAT3 antibody (mouse; #9139; Cell Signaling Technology), p-STAT3 antibody (rabbit; #9145; Cell Signaling Technology, Danvers, MA, USA), and GAPDH antibody (rabbit; #GB11002; Servicebio, Wuhan, China) at 4 °C overnight. The membranes were visualized using the ECL detection system (SigmaAldrich, Darmstadt, Germany), as described previously (Man et al., 2019).
4
ChIP Assay in Liver Cancer Cells
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ChIP assays were conducted in Huh7 and MHCC97‐H cells as the manufacturers instructed (Millipore Corp., Billerica, MA, USA). A STAT3 antibody (Cell Signal Technology, Danvers, MA, USA) was used to perform the immunoprecipitation experiment. PCR primer sequences for the DNA fragments targeting the FOXM1 and GLUT1 promoters are listed in Table S3 .
5
Investigating TLX3-STAT3 Interaction in HCC
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Protein expression was assessed in samples from both HCC cell lines and resected patient tissues by Western immunoblotting as described previously 38 (link). The primary antibodies used were: TLX3 (ab184011; Abcam), p-STAT3 (9145; Cell Signaling Technology), STAT3 (9139; Cell Signaling Technology), SNAI1(3895; Cell Signaling Technology), E-cadherin (14472; Cell Signaling Technology), N-cadherin (4061; Cell Signaling Technology), Vimentin (5741; Cell Signaling Technology) and β-actin (8457; Cell Signaling Technology). All protein expression was normalized toβ-actin after densitometric scanning.
Co-immunoprecipitation (CO-IP) was conducted to determine whether TLX3 protein was bound with STAT3 protein according to the protocol reported previously 37 (link). Briefly, HCC cells were lysed with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM ethylenediaminetetraacetic acid, 10 mM sodium butyrate) containing protease inhibitors. The cell lysate was incubated with the antibody against TLX3 (ab184011; Abcam) overnight at 4˚C and protein A/G-agarose beads were added. The mixture was shaken overnight at 4˚C and then rinsed with immunoprecipitation buffer. The supernatant was analyzed by Western immunoblotting with the STAT3 antibody (9139; Cell Signaling Technology).
Co-immunoprecipitation (CO-IP) was conducted to determine whether TLX3 protein was bound with STAT3 protein according to the protocol reported previously 37 (link). Briefly, HCC cells were lysed with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM ethylenediaminetetraacetic acid, 10 mM sodium butyrate) containing protease inhibitors. The cell lysate was incubated with the antibody against TLX3 (ab184011; Abcam) overnight at 4˚C and protein A/G-agarose beads were added. The mixture was shaken overnight at 4˚C and then rinsed with immunoprecipitation buffer. The supernatant was analyzed by Western immunoblotting with the STAT3 antibody (9139; Cell Signaling Technology).
6
Immunoblotting for STAT3 in Mouse Organs
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STAT3 antibody was purchased from Cell Signaling [9 9139]. Lysates from various mouse organs were prepared by snap-freezing in liquid nitrogen followed by homogenization with a small mortar and pestle in ice-cold RIPA buffer supplemented with protease inhibitors. Protein concentration was quantified using the BCA protein assay kit (Thermo Scientific, cat# 23225). Western blotting was performed as previously described [40] (link).
7
Protein Immunoprecipitation and Western Blot
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Total protein in TE-13 was harvested as input. The supernatants were incubated with 30 μl protein A/G agarose (Santa Cruz Biotechnology, USA) and 6 μl S1PR1 antibody (Abcam, USA) or 6 μl STAT3 antibody (Cell Signaling Technology, USA) at 4 °C overnight. Then, deposited immune complexes were analyzed by Western blotting using antibodies against S1PR1 and STAT3.
8
Cytokine Signaling in Murine B Cells
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For cytokine stimulation assays with murine B lymphocytes, cells were seeded at a density of 2.5×106/mL in 96-well plates, with a well solution volume of 200 µl. Cells were starved in serum-free medium for 2 hours, then were stimulated with either 0.1 nM of GM-CSF, 0.1 nM of IL-15, or both cytokines, or 0.1 nM of GIFT15 for 1 h at 37°C. Cells were collected, washed once with ice-cold PBS, and lysed in buffer supplemented with protease inhibitors and phosphatase inhibitor. Cell lysates were separated by SDS-PAGE, and Western blot analysis was performed with α-phosphorylated STAT3 and STAT5 antibodies (Cell Signaling, Danvers, MA). STAT3 antibody (Cell Signaling) detection was used as a loading control. Blotted proteins were visualized using enhanced chemiluminescence (ECL; Amersham, Amersham, UK). For detection of multiple STAT family members, the membranes were stripped of bound antibody and reprobed with antisera. For the quantification and identity of the recombinant GIFT15 protein, mouse IL-15 and GM-CSF antibodies were used (R&D Systems, Minneapolis, MN) and recombinant mouse IL-15 and/or mouse GM-CSF proteins were used as standards (R&D Systems, Minneapolis, MN).
9
Western Blot Analysis of IL-32, STAT3, and Bax
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After IR, the RIPA lysis buffer was used to lyse the cells. After the protein concentration was confirmed by the BCA protein detection kit (Beyotime, Shanghai, China), the proteins of the same concentration were separated using 10% SDS-PAGE and then transferred to PVDF membrane (Millipore, USA). Following the blocking step using 5% milk, antibodies were added to the membranes overnight at 4°C, including IL-32 antibody from Proteintech, USA, and STAT3 antibody, p-STAT3 antibody, Bax antibody, and GAPDH antibody from Cell Signaling Technology, USA. Subsequently, the membranes were incubated with the secondary antibodies (Cell Signaling Technology, USA) at room temperature for 2 hours. Immunoblotting protein was detected by enhanced chemiluminescence.
10
Western Blot Analysis of STAT3 Signaling
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Western blot experiments were performed as previously reported (Xue et al., 2017 (link); Qin et al., 2018 (link)). In brief, 3 × 105 cells were cultured in 6-cm dishes treated with specific concentrations of SDL-1 (0, 10, 20, and 40 μM) for 24 h. And then all cells were lysed in RIPA lysis buffer (Absin Bioscience Inc., Shanghai, China) containing protease inhibitor mixture (PMSF). The cell lysates were quantified by the BCA Protein Assay Kit (Absin, Shanghai, China). Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, United States). Block the membranes in 5% skim milk at room temperature and incubated with primary antibody overnight at 4 °C followed by incubation with appropriate secondary antibody at room temperature. Antibodies used are as follows: STAT3 antibody (Cell Signaling Technology, #12640S), p-STAT3 antibody (Cell Signaling Technology, #49081S), and GADPH antibody (Proteintech, #60004-1-Ig). The protein bands were detected by ECL Chemiluminescent Substrate Reagent Kit (Biosharp, Hefei, China) and scanned using the ImageQuant 800 (Amersham, United Kingdom).
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