IHC and immunoblotting were performed as we previously described 10 (link), 68 (link). Antibodies for IHC included p-STAT3 (Y705) antibody (Cell Signaling; #9145; 1:50), and rabbit polyclonal GLI1 and tGLI1 antibodies that we developed and validated 10 (link), 26 , 68 (link). Antibodies for immunoblotting included p-STAT3 (Y705) antibody (Cell Signaling, 1:1,000), STAT3 rabbit polyclonal antibody (Santa Cruz; C20; 1:1,000), α-tubulin and β-actin mouse monoclonal antibodies (Sigma), and rabbit polyclonal GLI1/tGLI1 antibody (Cell Signaling, 1:1,000). For IP, cells were transfected with flag-tagged GLI1, tGLI1 or STAT3 mutants, stimulated with 100 ng/ml EGF and 100 ng/ml SHH for 2 hrs, washed, fractionated, and precleared with protein G-agarose (Sigma; A2220). Precleared lysates were incubated with anti-flag M2 affinity gel or mouse IgG at 4°C overnight with gentle agitation. Pellets were collected, washed and subjected to SDS-PAGE and WB analysis. STAT3 IP was performed using antibodies from Santa Cruz (C-20). GLI1 and tGLI1 immunoprecipitation was performed using a GLI1 antibody (Santa Cruz, H-300) that recognizes the COOH-terminal end and, therefore, pulls down GLI1 and tGLI1.
P stat3 y705 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The P-STAT3 (Y705) antibody is a research-use only product that specifically detects the phosphorylated form of STAT3 at tyrosine residue 705. It is a fundamental tool for studying the activation of the STAT3 signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Protein g agarose, by Merck Group (2 mentions)
Stat3 rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
α tubulin and β actin mouse monoclonal antibodies, by Merck Group (2 mentions)
Rabbit polyclonal gli1 tgli1 antibody, by Cell Signaling Technology (2 mentions)
As 1109, by Aspen (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Eclipse ni, by Nikon (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Secondary antibody conjugated to alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Vectashield antifade containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
P s6k1, by Cell Signaling Technology (1 mentions)
Confocal spectral microscope tcs sp8 confocal microscope, by Leica (1 mentions)
6 protocols using p stat3 y705 antibody
1
Characterizing STAT3 and GLI1 Interactions
2
Characterizing STAT3 and GLI1 Interactions
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IHC and immunoblotting were performed as we previously described 10 (link), 68 (link). Antibodies for IHC included p-STAT3 (Y705) antibody (Cell Signaling; #9145; 1:50), and rabbit polyclonal GLI1 and tGLI1 antibodies that we developed and validated 10 (link), 26 , 68 (link). Antibodies for immunoblotting included p-STAT3 (Y705) antibody (Cell Signaling, 1:1,000), STAT3 rabbit polyclonal antibody (Santa Cruz; C20; 1:1,000), α-tubulin and β-actin mouse monoclonal antibodies (Sigma), and rabbit polyclonal GLI1/tGLI1 antibody (Cell Signaling, 1:1,000). For IP, cells were transfected with flag-tagged GLI1, tGLI1 or STAT3 mutants, stimulated with 100 ng/ml EGF and 100 ng/ml SHH for 2 hrs, washed, fractionated, and precleared with protein G-agarose (Sigma; A2220). Precleared lysates were incubated with anti-flag M2 affinity gel or mouse IgG at 4°C overnight with gentle agitation. Pellets were collected, washed and subjected to SDS-PAGE and WB analysis. STAT3 IP was performed using antibodies from Santa Cruz (C-20). GLI1 and tGLI1 immunoprecipitation was performed using a GLI1 antibody (Santa Cruz, H-300) that recognizes the COOH-terminal end and, therefore, pulls down GLI1 and tGLI1.
3
Quantifying ZO-1 and p-STAT3 in Pancreatic Tissue
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After the paraffin sections of the pancreas were deparaffinized, washed, and blocked, ZO-1 antibody (1:1,000; catalog no. 21773-1-AP, Proteintech, Wuhan, China) was added to it and incubated at 4°C. MPAC were incubated with p-STAT3Y705 antibody (1:100; catalog no. #9145, Cell Signaling Technology) at 4°C. After washing with PBS, CY3-labeled goat anti-rabbit antibody (1:50; catalog no. AS-1109, Aspen, Wuhan, China) was added, and the nuclei were stained with 4,6-diamino-2-phenylindole (DAPI, AS1075, Aspen, Wuhan, China). To evaluate the expression level of ZO-1 and p-STAT3, ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to measure the fluorescence intensity of ZO-1 and p-STAT3, and the fluorescence intensity was quantified under a high-power field.
4
Activation of STAT3 signaling in airway epithelial cells
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BEAS-2B and NHBE were seeded in 6 well plates at 3.0 × 105 cell/well and allowed to grow near 70% confluence in their growth media (BEBM). Cells were then treated with IFNβ (30 U/ml) for 24 h. After 24 h cells were treated with IL-22 (20 ng/ml) for 15 min then isolated in RIPA buffer (Thermo Scientific, Grand Island, NY) with Protease and Phosphatase inhibitors (Thermo Scientific, Grand Island, NY). Cells were scraped then sonicated at 15% in their respective tubes for one 15 s burst. Samples were spun down at 14,000 g for 15 min and supernatant transferred to new respective tubes. Protein was quantified and normalized via Bradford assay and loaded onto 4–12% NuPAGE Bis-Tris gel at 15μg of protein per sample. Transfers were done on iBlot (Invitrogen, Carlsbad, CA). pSTAT3 (Y705) antibody (Cell Signaling Technologies, Danvers, MA) was used to detect classically activated phosphorylated STAT3.
5
Quantifying Activated STAT3 in Hypothalamus
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The hypothalamus sections were fixed in 10% formalin (10 min), washed in PBS (5 × 8 min), and then incubated with 1% H2O2 and 0.3% NaOH (20 min), 0.3% glycine (10 min), and 0.03% sodium dodecyl sulfate (SDS; 10 min). After rinsing in PBS, the sections were blocked with 10% normal goat serum (in 0.3% Triton X-100; 60 min) and incubated overnight at 4 °C with the primary p-STAT3 (Y705) antibody (Cell Signaling Technology, Boston, MA, USA), diluted 1:300 in a block solution. After thorough rinsing in PBS (3 × 8 min), the sections were incubated with secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:600 in block solution overnight at 4 °C. Then, the preparations were washed in PBS (3 × 8 min) and mounted in Vectashield mounting medium with DAPI (Vector Labs, Burlingame, CA, USA) under a cover glass. The sections were viewed using an inverted microscope Eclipse Ni (Nikon, Tokyo, Japan), and pictures were taken. The percentage of p-STAT3-positive nuclei to the total number of nuclei was calculated using ImageJ software (NIH).
6
Immunofluorescence Analysis of P-STAT3 and P-S6K1
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Cells were grown on coverslips housed in 24-well plates. Once cells were treated as indicated, they were washed with ice-cold phosphate buffered saline (PBS) and fixed using methanol. Cells were blocked with 10% BSA + 0.1% saponin in PBS for 1 hr. The cells were then incubated overnight at room temperature with P-STAT3 (Y705) antibody (1:250; Cell Signaling) and P-S6K1 (1:350; Cell Signaling). The excess antibody was washed off using three 15-min washes with PBS and then the coverslips were incubated with secondary antibody (1:500) for 1 hr followed by three more washes with PBS. The coverslips were then mounted using VECTASHIELD Antifade containing 4,6-diamidino-2-phenylindole (DAPI) Vector Laboratories, Burlingame, USA). Slides were imaged via Confocal Spectral Microscope (TCS-SP8 confocal microscope, Leica, Germany).
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