Paraffin-embedded clinical NSCLC samples and metastatic lung tumors in mouse models were subjected to IHC staining as previously described [21 (link)]. For the detection of FXR, IL-6, IL6ST and p-STAT3, the following primary antibodies were used: anti-bile acid receptor NR1H4 (1:100; cat. no. ab187735; Abcam, Cambridge, UK), anti-IL-6 (1:100; cat. no. ab9324; Abcam), anti-CD130 (gp130) (1:100; cat. no. ab227058; Abcam) and anti-Phospho-STAT3 (Tyr705) (1:200; cat. no. 9145s; Cell Signaling Technology, Inc., Beverly, MA). Concentration-matched non-specific mouse or rabbit IgG served as isotype controls. The IHC staining results were scored independently and blindly by two skilled pathologists, and a final consensus was reached. The staining intensity of tumor cells was scored as negative (0), weak (1), medium (2), and strong (3), respectively. The percentage of positive cells was scored as follows: 0% (0), 1%–25% (1), 26%–50% (2), 51%–75% (3), and 76%–100% (4), respectively. The final IHC scores of human FXR, IL-6, IL6ST and p-STAT3 were obtained by multiplying the staining intensity score with the positive-cell percentage score, and stratified as follows: Low, score < 6 or high, score ≥ 6, in line with our previous studies [18 (link), 19 (link)].
Anti phospho stat3 tyr705
Manufactured by Abcam
Sourced in United Kingdom
Anti-Phospho-STAT3 (Tyr705) is a laboratory antibody product that specifically recognizes the tyrosine 705 phosphorylated form of the STAT3 protein. This antibody can be used to detect and measure the levels of activated STAT3 in various experimental systems.
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Lab products found in correlation
Anti il 6, by Abcam (2 mentions)
Anti stat3, by Abcam (2 mentions)
Anti cd130 gp130, by Abcam (1 mentions)
Anti lc3b, by Merck Group (1 mentions)
Hrp goat anti rabbit igg, by Proteintech (1 mentions)
Anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
West femto substrate trial kit, by Thermo Fisher Scientific (1 mentions)
Anti beclin 1, by Proteintech (1 mentions)
Goat anti mouse igg hrp, by Proteintech (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Anti p38 mapk, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Anti crp, by Abcam (1 mentions)
Bca protein determination kit, by Thermo Fisher Scientific (1 mentions)
Anti total stat3, by Abcam (1 mentions)
Anti phosho akt ser473, by Cell Signaling Technology (1 mentions)
Anti total erk, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
5 protocols using anti phospho stat3 tyr705
1
Immunohistochemical Analysis of NSCLC Samples
2
Protein Expression and Activation Analysis
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The cells were lysed in RIPA lysis buffer (Millipore) containing protease (Bimake) and phosphatase (Bimake) inhibitors. The samples were resolved by SDS-PAGE and transferred to PVDF membrane after blocking, the membranes were incubated with primary antibodies overnight at 4 °C and then exposed to secondary antibodies. Protein bands were visualized using West Femto Substrate Trial Kit (Thermo Scientific). The primary antibodies included anti-p38 MAPK (Cell Signaling Technology), anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology), anti-STAT3 (Abcam), anti-phospho-STAT3 (Tyr705) (Abcam), anti-LC3B (Sigma), anti-Beclin1 (proteintech), anti-β-Tubulin (Cell Signaling Technology), and anti-GAPDH (proteintech). The secondary antibodies were goat anti-mouse IgG HRP (proteintech) and goat anti-rabbit IgG HRP (proteintech).
3
Protein Extraction and Western Blot Analysis
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Proteins from frozen pNEN tissue specimen (60–80 mg) pulverized in liquid nitrogen or from BON1 cells pellets were extracted using RIPA buffer containing complete protease inhibitors (Sigma-Aldrich) and supplemented with phosphatase inhibitor sets 1 and 2 (Sigma-Aldrich). Using an ultrasonic homogenizer (SonoPuls mini20 Bandelin®, Berlin, Germany), the suspensions were subjected to a 30-s sonication step on ice (ampl. 80%, 0.99 kJ) and subsequently centrifuged at 16,000 g for 10 min.
Supernatants were collected and divided into aliquots, and the total protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions.
Western blot was performed as previously described (37 (link)) using the following monoclonal antibodies: anti-CRP, anti-IL-6, anti-total-STAT3 (all Abcam), anti-phospho-STAT3 (Tyr705), anti-total-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-total-AKT and anti-phosho-AKT (Ser473) (all Cell Signaling). The secondary antibody used was goat anti-rabbit (R&D Systems). Quantification of protein expression was performed using ImageJ (EHD imaging, Damme, Germany).
Supernatants were collected and divided into aliquots, and the total protein concentration was determined using a BCA Protein Determination Kit (Thermo Scientific) following the manufacturer’s instructions.
Western blot was performed as previously described (37 (link)) using the following monoclonal antibodies: anti-CRP, anti-IL-6, anti-total-STAT3 (all Abcam), anti-phospho-STAT3 (Tyr705), anti-total-ERK, anti-phospho-ERK (Thr202/Tyr204), anti-total-AKT and anti-phosho-AKT (Ser473) (all Cell Signaling). The secondary antibody used was goat anti-rabbit (R&D Systems). Quantification of protein expression was performed using ImageJ (EHD imaging, Damme, Germany).
4
STAT3 Protein Expression and Phosphorylation
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Cancer cells were lysed with the lysis buffer. Protein samples were subjected to SDS-polyacrylamide gels electrophoresis. The gels were transferred onto PVDF membranes (Millipore). Cell proteins were extracted and subjected to Western blot analysis using anti-STAT3 (Cat. 9139S) (Cell Signalling Technology), anti-phospho-STAT3-Tyr705 (Cat. 76,315) and anti- β-actin (Cat. ab6276) (Abcam).
5
Quantifying STAT3 Activation in Cells
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Cells were seeded in a 6-well plate at a density of 1 × 106 cells/well in 2 mL media and cultured overnight. Then the cells were treated with TTF1-NP (50, 100 and 200 μM) or DMEM (Vehicle) after 48 h. Briefly, the cells were washed and blocked for 20 min in 3% hydrogen peroxide. The cells were incubated with primary antibodies overnight at 4 °C and then with horseradish peroxidase-conjugated secondary antibodies for 30 min at 37 °C. The cells were then developed with 3,3’-diaminobenzidine and counter-stained with hematoxylin. The cells were photographed by a light microscope (Olympus, Japan) at 200× magnification, three different fields were chosen randomly in each well. The following antibodies were used: anti-STAT3 (1:200 dilution; Abcam, Cambridge, UK), anti- phospho-STAT3 (Tyr705) (1:500 dilution; Abcam).
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