The rat livers and AML12 cells were lysed by RIPA buffer, separated by 10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane. After blocking in 5% Bovine Serum Albumin (BSA), the membrane was incubated with the phospho-JAK2 (Tyr931) (1:1,000, affinity), total JAK2 (1:1,000, proteintech), phospho-STAT3 (Tyr705) (1:1,500, abcam), total STAT3 (1:2000, abcam), IL-6 (1:500, bioworld), IL-18 (1:2000, bioworld), IL-1β (1:500, sino biological), TNFα (1:1,000, sino biological), HIF-1a (1:1,000, Novusbio), HIF-2a (1:1,000, Cell Signaling Technology), VEGFa (1:1,000, Novusbio) primary antibody seperately overnight at 4°C, and then was incubated with HRP conjugated secondary antibody for 1 h at room temperature. Western blots were displayed by advanced enhanced chemiluminescence kit and then semi-quantified by Clinx (Shanghai Qinxiang Scientific Instrument Co., Ltd).
Total stat3
Manufactured by Abcam
Sourced in United Kingdom
Total STAT3 is a quantitative colorimetric assay that measures the total amount of STAT3 protein in cell and tissue lysates. It provides a direct and specific measure of STAT3 levels without the need for Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Sino Biological (1 mentions)
Phospho jak2, by Proteintech (1 mentions)
Tnf α, by Sino Biological (1 mentions)
Vegfa, by Novus Biologicals (1 mentions)
Phospho stat3 tyr705, by Abcam (1 mentions)
Interleukin 6 (il 6), by Bioworld Technology (1 mentions)
Hif 1a, by Novus Biologicals (1 mentions)
Hif2a, by Cell Signaling Technology (1 mentions)
Total akt, by Abcam (1 mentions)
Phosphorylated akt, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Phosphorylated stat3, by Abcam (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Anti mouse igg secondary antibody, by Merck Group (1 mentions)
P stat3 ser727, by Abcam (1 mentions)
3 protocols using total stat3
1
Signaling Pathways in Liver and AML12 Cells
2
Protein Expression Analysis of A498 and Renca Cells
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The A498 cells and Renca cells were collected for protein samples with NP40 solution. 25 μg protein samples were separated by SDS-PAGE, followed with transferring to PVDF membranes. Next, samples were examined by immunoblotting with primary antibodies against: α-SMA (1:1000, Abcam, Cambridge, UK), AhR (1:5000, Abcam, Cambridge, UK), phosphorylated-Src (1:500, Abcam, Cambridge, UK), total Src (1:500, Abcam, Cambridge, UK), phosphorylated AKT (1:500, Abcam, Cambridge, UK), total AKT (1:500, Abcam, Cambridge, UK), phosphorylated STAT3 (1:500 Abcam, Cambridge, UK), total STAT3 (Abcam, Cambridge, UK) and β-actin (1:1000, Abcam, Cambridge, UK) respectively at 4°C overnight. Then HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK) was incubated for 1 hour at room temperature, and visualized by using ECL detection kit (Thermo, MA, USA).
3
Protein Extraction and Western Blot Analysis
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Whole-cell protein was isolated as described previously [7] . Briefly, for tissue protein extraction, the tumor tissues were homogenized in ice-cold RIPA (radioimmunoprecipitation assay) lysis buffer (Millipore). The tissue lysates were incubated at 4°C for 1 hour with rotation followed by clarification of tissue debris by centrifugation at 12, 000 rpm for 10 minutes. The protein concentration of tumor extracts was determined using the BCA Protein Assay Kit (Pierce). Western blotting was performed as previously described [7] . Briefly, The following antibodies to E-cadherin (Cell Signaling), vimentin (Cell Signaling), snail (Cell Signaling), slug (Cell Signaling), ZO-1 (Cell Signaling), N-cadherin (Cell Signaling), claudin-1 (Cell Signaling), β-actin (Sigma Aldrich), total STAT3 (Abcam), pSTAT3 (Ser727) (Abcam), pSTAT3 (Tyr705) (Abcam), pSTAT (Tyr694) (Cell Signaling), total AKT (Cell Signaling), pAKT (Ser473) (Cell Signaling), pAKT (Thr308) (Cell Signaling), and pERK (Cell Signaling) were applied for protein detection. Antibody binding was revealed using an HRPconjugated anti-rabbit IgG or anti-mouse IgG secondary antibody (Sigma). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millopore) and exposure to Tanon 6200 Luminescent Imaging Workstation (Tanon Science & Technology Co., Ltd., Shanghai, China).
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