Tissues were homogenized in Mammalian protein extraction reagent (M-PER, ThermoScientific, Waltham, MA, USA). Western blots were run as previously described [13 ]. Briefly 10 μg of protein was run on a 10% BisTris gel. Primary antibodies were incubated overnight (phosphoSTAT3 #9145, and SOCS3 #2923 Cell signaling, Danvers, MA, USA; TNF-alpha (ab6671) and MyD88 (ab2064) Abcam, Cambridge, MA, USA; TLR4 (img-579A) Imgenex San Diego CA, USA; TLR4-MD2 (14-9924-81) eBioscience San Diego CA, USA). GAPDH was used as a loading control (#2118, Cell Signaling). Secondary antibody was incubated 1 hour (Anti-rabbit IgG HRP-linked, Cell Signaling). The membrane was exposed to film for 5–45 minutes and developed in a 100 Plus automatic X-Ray film Processor (All Pro Imaging, Hicksville, NJ, USA). The film was analyzed by Imagequant v 5.1 software (Biosciences, Amersham, UK).
Phospho stat3 9145
Manufactured by Cell Signaling Technology
Sourced in United States
The Phospho-STAT3 (9145) product is a rabbit monoclonal antibody that detects endogenous levels of STAT3 protein only when phosphorylated at tyrosine 705. This antibody can be used in techniques such as western blotting to analyze the phosphorylation status of STAT3.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human bdnf, by Thermo Fisher Scientific (2 mentions)
Twist, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
Normal human fibroblasts detroit 551, by American Type Culture Collection (2 mentions)
Trka sc 14024, by Santa Cruz Biotechnology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Vector elite abc kit, by Vector Laboratories (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Phospho creb 9198, by Cell Signaling Technology (2 mentions)
Tfeb pa5 96632, by Thermo Fisher Scientific (2 mentions)
Histoclear, by National Diagnostics (2 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Bca protein quantification assay, by Beyotime (1 mentions)
Ki 67 ncl ki67p, by Leica (1 mentions)
7 protocols using phospho stat3 9145
1
Western Blot Analysis of Inflammatory Signaling
2
Characterization of Head and Neck Cancer Cell Lines
3
Protein Quantification and Western Blot Analysis
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Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer and phosphatase inhibitors. The protein concentration was measured using the bicinchoninic acid (BCA)-protein quantification assay (Beyotime Biotechnology, Shanghai, China). Normalized lysates (30 μg) were separated by electrophoresis on 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane (Millipore Corporation). The membrane was blocked for 1 h at room temperature and incubated overnight at 4°C with primary antibody. After three washes with TBST, the membrane was incubated with horseradish peroxidase-(HRP)-conjugated IgG. Signals were visualized with enhanced chemiluminescence (ECL; Millipore Corporation). Primary antibodies against Smad3 (#9523), phospho-Stat3 (#9145), Stat3 (#12640), JAK1 (#3344), JAK2 (#3230) (dilution, 1:1,000; Cell Signaling Technology, Beverly, MA, USA), phospho-Smad3 (ab52903), phospho-JAK2 (ab32101) (dilution, 1:1,000; Abcam, Cambridge, MA, USA), phospho-JAK1 (sc-101716; dilution, 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used.
4
Immunohistochemical Analysis of Tissues
5
Characterization of Head and Neck Cancer Cell Lines
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The head and neck squamous cell carcinoma cell lines OSC19, UMSCC1, Tu138, FADU, UMSCC-22A cells were maintained as described previously [28 (link), 29 (link)]. The OSC19-Luc cell line is a stable transfectant constitutively expressing luciferase and GFP [30 (link)]. Normal human fibroblasts (Detroit 551) were purchased from ATCC. Recombinant human BDNF and EGF were obtained from Peprotech (Rocky Hill, NJ). The following antibodies were utilized: TrkA (sc-14024, Santa Cruz Biotechnology, Inc.), TrkB (sc-8316), TrkC (sc-117), NGF (sc-548), BDNF (sc-546), vimentin (sc-51719), GAPDH, MAPK (9107, Cell Signaling, Inc.), phospho-MAPK (9101), STAT3 (9132), phospho-STAT3 (9145), AKT (9272), phospho-AKTser473 (3787), Twist (4119), E-cadherin (4065), N-cadherin (4061), β-catenin (9562), PCNA (2586), Slug, Snail and LYVE-1.
6
Immunohistochemical Analysis of Signaling Pathways
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Paraffin-embedded tissue sections were cleared with histoclear (National Diagnostics) and graded alcohol using standard techniques. Immunohistochemistry was performed using standard techniques [56 (link)]. Antigen retrieval was accomplished by heating sections in 7 mM or 10 mM citrate, under pressure. Sections were incubated with primary antibody overnight at 4 °C. Staining was detected using Vector Elite ABC Kits (Vector Laboratories, Burlingame, CA, USA) and 3,3-diaminobenzidine as chromogen (Vector Laboratories). Primary antibodies were against: phospho-STAT3 (9145), phospho-STAT5 (9314), CREB (9197), phospho-CREB (9198), and NFAT (5861), all from cell signaling (Danvers, MA) as well as TFEB (PA5-96632) from Invitrogen (Grand Island, NY).
7
Immunohistochemical Analysis of Signaling Pathways
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Paraffin-embedded tissue sections were cleared with histoclear (National Diagnostics) and graded alcohol using standard techniques. Immunohistochemistry was performed using standard techniques 59 (link). Antigen retrieval was accomplished by heating sections in 7 mM or 10 mM citrate, under pressure. Sections were incubated with primary antibody overnight at 4°C. Staining was detected using Vector Elite ABC Kits (Vector Laboratories, Burlingame, CA, USA) and 3,3-diaminobenzidine as chromogen (Vector Laboratories). Primary antibodies were against: phospho-STAT3 (9145), phospho-STAT5 (9314), CREB (9197), phospho-CREB (9198), and NFAT (5861), all from cell signaling (Danvers, MA) as well as TFEB (PA5-96632) from Invitrogen (Grand Island, NY).
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