Total protein was extracted from the cells subjected to the different treatments using RIPA buffer (Beyotime Institute of Biotechnology) at 4℃. After resolution of the total proteins by electrophoresis, the protein bands were electrotransferred to polyvinylidene difluoride membranes for incubation with primary antibodies against the following target proteins: GAPDH (1:5000 dilution, ab8245, Abcam), caspase‐3 (1:1000, ab4051, Abcam), caspase‐9 (1:1000, ab25758, Abcam), cleaved caspase‐3 (1:1000, ab2302, Abcam), cleaved caspase‐9 (1:1000, ab2324, Abcam), Src homology region 2 domain‐containing phosphatase 1 (SHP‐1) (1:2000, ab227503, Abcam), signal transducer and activator of transcription 3 (STAT3) (1:2500, ab31370, Abcam), and phosphor STAT3 (1:500, ab7315, Abcam). Thereafter, the membranes were incubated with goat anti‐rat secondary antibody (1:1000, A0216, Beyotime Institute of Biotechnology) and horseradish peroxidase‐conjugated goat anti‐rabbit antibody (1:1000; A0208, Beyotime Institute of Biotechnology). The immune response zone was finally detected by development with a microporous chemiluminescence western blot kit (Millipore Sigma).
Ab31370
Manufactured by Abcam
Sourced in United Kingdom
Ab31370 is a laboratory equipment product offered by Abcam. It is a protein that functions as a molecular marker. The core function of this product is to serve as a detection tool in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab7315, by Abcam (2 mentions)
Ab4051, by Abcam (2 mentions)
Ab227503, by Abcam (2 mentions)
Ab2324, by Abcam (2 mentions)
Ab25758, by Abcam (2 mentions)
A0216, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
A0208, by Beyotime (2 mentions)
Ab195055, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab86430, by Abcam (1 mentions)
High efficiency ripa lysis buffer, by Solarbio (1 mentions)
Bca kit, by Yeasen (1 mentions)
Ab39636, by Abcam (1 mentions)
Ab7970, by Abcam (1 mentions)
4 protocols using ab31370
1
Western Blot Analysis of Apoptosis Markers
2
Western Blot Analysis of JAK2/STAT3 Signaling
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Total protein of cells was extracted using high-efficiency RIPA lysis buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in strict accordance with the instructions. The protein concentration was determined using a BCA kit (20201 ES76, Yeasen Company, Shanghai, China). Then, the protein was separated by polyacrylamide gel electrophoresis and then transferred to PVDF membranes by wet transfer method. Next, the membranes were blocked using 5% BSA at room temperature for 1 h and probed overnight at 4 °C with the following diluted primary antibodies: mouse anti-human GAPDH (ab9425, 1:2500, Abcam, Cambridge, UK), JAK2 (ab39636, 1:1000, Abcam, Cambridge, UK), p-JAK2 (ab195055, 1:1000, Abcam, Cambridge, UK), STAT3 (ab31370, 1:500, Abcam, Cambridge, UK), and p-STAT3 (ab86430, 1:500, Abcam, Cambridge, UK). The membranes were washed for three times (5 min/time) with Tris-buffered saline Tween-20 (TBST), and horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:20,000, Abcam, Cambridge, UK) was added for 1 h of incubation at room temperature. Then, ImageJ 1.48u software (National Institutes of Health) was performed for protein quantification analysis. The ratio of the gray value of the target band to GAPDH was representative of the relative protein expression. The experiment was repeated three times.
3
Western Blotting of Apoptosis Markers
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Extracts of cells and specimens were prepared in RIPA buffer (Beyotime Biotechnology) at 4 °C. WB was performed with the cell extracts using commercially available primary antibodies. Primary antibodies used in this study includes: Antibodies to GAPDH (1:5000, ab8245, Abcam), Caspase-3 (1:1000, ab4051, Abcam), Caspase-9 (1:1000, ab25758, Abcam), cleaved Caspase-3 (1:1000, ab2302, Abcam), cleaved Caspase-9 (1:1000, ab2324, Abcam), SHP-1 (1:2000, AB227503, Abcam), STAT3 (1:2500, ab31370, Abcam), and phosphor STAT3 (1:500, ab7315, Abcam). A combination of horseradish peroxidase-conjugated goat anti-rabbit (1:1,000; A0208) and goat anti-mouse secondary antibodies (1:1000; A0216, all from Beyotime Biotechnology) were used to detect the immunoreactive bands; development was performed using the Millipore chromogenic chemiluminescence detection kit (Millipore Sigma, Burlington, MA).
4
Protein Expression Analysis in Bladder Cancer
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Total proteins in cultured bladder cancer cells were prepared by lysis using the Cell Total Protein Lysis kit (Sangon Biotech, Shanghai, China), following the producer’s instructions. The protein concentration was detected using the BCA method. Then, the protein was boiled at 100 °C for 5 min, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane pre-immersed with methanol. Following blocking in 5% bovine serum albumin solution for 2 h at room temperature, PVDF membranes were incubated with primary antibodies at 4 °C overnight and with secondary antibodies for 1–2 h at room temperature. The relative protein levels were finally detected by development with enhanced ECL substrate solutions (#32106; Thermo Fisher Scientific). The bands were observed using Tanon-5200CE (Biotanon, Shanghai, China). The expression of GAPDH protein was simultaneously analyzed as the internal standard for non-phosphorylation proteins. Primary antibodies used in Western blotting include anti-ITGA2 (#BM5058; Boster; 1:1000), anti-p-AKT (#4060S; CST; 1:1000), anti-AKT (#2920S; CST; 1:1000), anti-p-p65 (#3033S; CST; 1:1000), anti-p65 (#ab7970; Abcam; 1:1000), anti-p-STAT3 (#9145; CST: 1:1000), anti-STAT3 (#ab31370; Abcam; 1:1000), and anti-GAPDH (#60004-1-Ig; Proteintech; 1:1000).
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