Tissues were lysed in radioimmunoprecipitation (RIPA) buffer and total protein concentration was determined with a bicinchoninic acid (BCA) assay (Beyotime). Then, 20 μg of total protein from each sample was separated by 10% SDS-PAGE and then transferred to PVDF membranes. Membranes were then washed, blocked and incubated sequentially with specific primary antibodies: rabbit polyclonal antibody LIF (1:1,000), LIFR (1:200); p-STAT3 (1:2,000), ER-α (1:1,000); gp130 (1:1,000); JAK1 (1:5,000) (Abcam) and mouse polyclonal anti-GAPDH (1:3,000; Santa Cruz Biotechnology). Incubation in primary antibodies was followed by incubation with goat anti-rabbit secondary antibody (1:5,000, Santa Cruz Biotechnology). Positive antibody binding reactions were detected by an enhanced chemiluminescence using an ECL system and each experiment was performed in triplicate. The relative densitometry of the band was measured using ImageJ software (Bethesda).
Gp130
Manufactured by Abcam
Sourced in United Kingdom, United States
Gp130 is a membrane glycoprotein that serves as a signal-transducing receptor subunit for several cytokines, including interleukin-6, interleukin-11, and ciliary neurotrophic factor. It plays a crucial role in the activation of the JAK-STAT signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P stat3, by Abcam (4 mentions)
Gp130, by Santa Cruz Biotechnology (3 mentions)
Nmda receptors, by Bioss Antibodies (3 mentions)
Nmda receptors, by Abcam (3 mentions)
Pstat3, by Bioss Antibodies (3 mentions)
Gad67, by Abcam (3 mentions)
Interleukin 6 (il 6), by Bioss Antibodies (3 mentions)
Interleukin 6 (il 6), by Abcam (3 mentions)
Gapdh, by Abcam (2 mentions)
Il 12rβ2, by Abcam (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Beyotime (1 mentions)
Pias3, by Abcam (1 mentions)
L ascorbic acid, by Cayman Chemical (1 mentions)
Activated caspase 3, by Abcam (1 mentions)
Bcl xl, by Abcam (1 mentions)
Mcl 1, by Abcam (1 mentions)
Socs3, by Abcam (1 mentions)
Bgc 823, by Keygene (1 mentions)
9 protocols using gp130
1
Protein Expression Analysis in Cell Lysates
2
Luteolin's Anti-Gastric Cancer Signaling
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Luteolin is purified and provided from Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine (Beijing, China), and the purity of the product was over 98%, detected by HPLC (UV). Gastric tumor cell lines of SGC7901, SGC7901/DDP, HGC27, MGC803, BGC803 and BGC823 were purchased from Keygene (Jiangsu, China). Monoclonal antibodies against HSP-90, STAT3 and phosphor-STAT3 (Tyr705), STAT1 and phosphor-STAT1 (Tyr701), Akt, phosphor-Akt (Ser473), Erk and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) were purchased from Cell Signaling Technology (Boston, MA, USA). Monoclonal antibodies against SHP-1, SHP-2, Bcl-xl, Survivin, Mcl-1, SOCS3, PIAS3, gp130, ki0–67, activated caspase-3 and GAPDH were purchased from Abcam (Burlingame, CA, USA). BCA Protein Assay Kit was purchased from Pierce (Rockford, IL, USA). l -Ascorbic acid was purchased from Cayman (Ann Arbor, MI, USA), α-Tocopherol was purchased from Selleckchem (Houston, TX, USA). DCFDA was purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Analyzing JAK-STAT Signaling in Gastric Cancer Cells
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Whole cell lysates were prepared from BGC823 or SGC-7901 cells treated as previous designed and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 100 V for 1 hour. Next, the separated proteins were transferred to NC membranes (Millipore, Billerica, MA). The samples were blocked with 5% bovine serum albumin (BSA) in TBS containing 0.1% Tween-20 for 1 hour at room temperature and incubated overnight at 4°C with one of the following antibodies: JAK (1:200, Abcam, Cambridge, UK), phospho JAK (Ser473) (1:200, Abcam), STAT3 (1:300, Abcam), phospho STAT3 (Tyr705) (1:300, Abcam), BCL-2 (1:200, Abcam), GP130 (1:300, Abcam) and actin (1:500, Abcam). The samples were washed with TBST and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Proteins were visualized by ECL western blotting reagent (Thermo Fisher).
4
Immunohistochemical Analysis of PVN Markers
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After the brain tissue was embedded in paraffin, the part between the optic chiasm and mammillary body was resected in the rostro-caudal direction. The tissue was serially sectioned on a paraffin slicer, and sections that were approximately 1.50 mm from the bregma were obtained [25 (link), 26 (link)]. After dewaxing the slices, 3% H2O2 was used to block endogenous peroxidase activity, and 0.01 M citric acid was used to retrieve the antigens prior to antibody incubation. Then, the slices were incubated with primary antibodies overnight at 4 °C [27 , 28 (link)]. The sections were immunohistochemically labelled to identify IL-6 (Bioss, China, 1:100), Gp130 (Santa Cruz, America, 1:20), pSTAT3 (Bioss, China, 1:50), NMDA receptors (Bioss, China, 1:50), and GAD67 (Abcam, England, 1:2000) and then incubated with secondary antibodies (anti-mouse for Gp130 and GAD67; anti-rabbit for NMDA receptors, pSTAT3 and IL-6) for 20 min at room temperature. For each rat, the positive neurons within the bilateral borders of the PVN were manually counted in three consecutive sections, and the average value is reported.
5
Immunohistochemical Evaluation of IL-35 in ICC
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Tissue microarrays were constructed according to a previous research report.25 Two different tissue microarray blocks extracted from each case (3‐mm‐diameter each) were used for TMA construction.
Monoclonal rabbit antihuman antibodies P35, EBI3, gp130 and IL‐12Rβ2 (1:100 dilution) were purchased from Abcam (Cambridge, UK). IHC was used to detect the expression of P35, EBI3, gp130 and IL‐12Rβ2 in tissues of ICC, and surgical produces were carried out as described.26 Two experienced observers were responsible for sliced evaluation in a double blind method. Expression of IL‐35, EBI3 gp130 and IL‐12Rβ2 was evaluated by a semiquantitative scoring system.27 The final score was made by positively stained tumor cells (0, no positive tumor cells; 1, <10%; 2, 10%‐35%; 3, 35%‐75%; 4, >75%) multiplied by staining intensity (1, no staining; 2, weak; 3, moderate; 4, strong). Eventually, total scores ≥8 were defined as high expression and those <8 were defined as low expression. Representative images of positive and negative controls for IHC are shown in Figure S1 .
Monoclonal rabbit antihuman antibodies P35, EBI3, gp130 and IL‐12Rβ2 (1:100 dilution) were purchased from Abcam (Cambridge, UK). IHC was used to detect the expression of P35, EBI3, gp130 and IL‐12Rβ2 in tissues of ICC, and surgical produces were carried out as described.
6
Western Blot Analysis of Immune Proteins
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Protein (30 μg) was separated by 10% SDS‐PAGE electrophoresis, then transferred to PVDF (Millipore, Temecula, CA, USA), and the membranes were blocked in 5% BSA for 1 hour, then incubated with primary antibodies (P35, EBI3, gp130 and IL‐12Rβ2 (1:1000 dilution; Abcam) at 4°C overnight. β‐Actin (Kangcheng, Shanghai, China) was used as an internal control. After 12 hours, the membranes were incubated with secondary antibody (Kangcheng) for 1 hour at room temperature. Finally, the protein bands were detected by enhanced chemiluminescent (ECL) substrate and processed by Image, Lab software (Bio‐Rad, Mississauga, ON, Canada).
7
Western Blot Analysis of Cell Signaling
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Cleaved Caspase 3 (9664, Cell Signaling Technology (CST), Danvers, MA, USA), Caspase 3 (9662, CST, MA, USA), Cyclin D1 (55506, CST, MA, USA), phospho-ERK1/2 (4370, CST, MA, USA), ERK1/2 (4695, CST, MA, USA), GAPDH (2118, CST, MA, USA), gp130 (ab202850, Abcam, UK), IL11 (X203, Aldevron, Germany), p-JNK (4668, CST, MA, USA), JNK (9252, CST, MA, USA), Ki67 (ab16667, Abcam, UK), PCNA (13110, CST, MA, USA), p-STAT3 (4113, CST, MA, USA), STAT3 (4904, CST, MA, USA), anti-mouse HRP (7076, CST, MA, USA), anti-rabbit HRP (7074, CST, MA, USA).
8
Immunohistochemical Analysis of PVN Markers
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After the brain tissue was embedded in para n, the part between the optic chiasm and mammillary body was resected in the rostro-caudal direction. The tissue was serially sectioned on a para n slicer, and sections that were approximately 1.50 mm from the bregma were obtained [25, 26] . After dewaxing the slices, 3% H 2 O 2 was used to block endogenous peroxidase activity, and 0.01 M citric acid was used to retrieve the antigens prior to antibody incubation. Then, the slices were incubated with primary antibodies overnight at 4°C [27, 28] . The sections were immunohistochemically labelled to identify IL-6 (Bioss, China, 1:100), Gp130 (Santa Cruz, America, 1:20), pSTAT3 (Bioss, China, 1:50), NMDA receptors (Bioss, China, 1:50), and GAD67 (Abcam, England, 1:2000) and then incubated with secondary antibodies (anti-mouse for Gp130 and GAD67; anti-rabbit for NMDA receptors, pSTAT3 and IL-6) for 20 minutes at room temperature. For each rat, the positive neurons within the bilateral borders of the PVN were manually counted in three consecutive sections, and the average value is reported.
9
Immunohistochemical Analysis of PVN Markers
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After the brain tissue was embedded in paraffin, the part between the optic chiasm and mammillary body was resected in the rostro-caudal direction. The tissue was serially sectioned on a paraffin slicer, and sections that were approximately 1.50 mm from the bregma were obtained [25, 26] . After dewaxing the slices, 3% H 2 O 2 was used to block endogenous peroxidase activity, and 0.01 M citric acid was used to retrieve the antigens prior to antibody incubation. Then, the slices were incubated with primary antibodies overnight at 4°C [27, 28] . The sections were immunohistochemically labelled to identify IL-6 (Bioss, China, 1:100), Gp130 (Santa Cruz, America, 1:20), pSTAT3 (Bioss, China, 1:50), NMDA receptors (Bioss, China, 1:50), and GAD67 (Abcam, England, 1:2000) and then incubated with secondary antibodies (anti-mouse for Gp130 and GAD67; anti-rabbit for NMDA receptors, pSTAT3 and IL-6) for 20 minutes at room temperature. For each rat, the positive neurons within the bilateral borders of the PVN were manually counted in three consecutive sections, and the average value is reported.
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