The immunoblotting assay was done as previously described41 (link). Primary antibodies used in this study were listed as follows: anti-eNOS (ab199956; Abcam), anti-nNOS (ab5586; Abcam), anti-iNOS (ab15323; Abcam), anti-biotin (7075 S; Cell Signaling Technology), anti-Raf-1 (ab137435; Abcam), anti-STAT3 (ab109085; Abcam), anti-p-STAT3 (Y705) (ab76315; Abcam), and anti-β-actin antibodies (#5125; Cell Signaling Technology).
Ab109085
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab109085 is an antibody that targets the protein Tropomyosin 1 (alpha chain). It is suitable for use in various research applications, including immunohistochemistry and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab76315, by Abcam (5 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab108596, by Abcam (2 mentions)
Ab32101, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab199956, by Abcam (1 mentions)
Ab137435, by Abcam (1 mentions)
Ab15323, by Abcam (1 mentions)
Ab19898, by Abcam (1 mentions)
Bicinchoninic acid kit, by Tiangen Biotech (1 mentions)
Protein lysis solution, by Tiangen Biotech (1 mentions)
Ab68141, by Abcam (1 mentions)
Ab170880, by Abcam (1 mentions)
Ab76242, by Abcam (1 mentions)
Ab77600, by Abcam (1 mentions)
Ab218695, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab214821, by Abcam (1 mentions)
8 protocols using ab109085
1
Immunoblotting Assay for Protein Expression
2
Western Blot Profiling of Cellular Proteins
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Total proteins in cells were extracted using protein lysis solution (Tiangen Biotech, Beijing, China). The protein concentration was measured with a bicinchoninic acid kit (Tiangen Biotech). The protein extracts were resolved through sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Bio-Rad, Berkeley, CA, USA), which were blocked with 5% non-fat milk solutions for 1 h at room temperature. The target protiens were then detected using primary antibodies against human TUSC3 (1:500; ab77600), signal transducer and activator of transcription 3 (STAT3; 1:1,000; ab109085), mouse double minute 2 homolog (MDM2; 1:500; ab170880), p53 (1:500; ab76242), MET (1:1,000; ab68141), CD133 (1:500; ab19898), zinc finger protein, X-linked (ZFX; 1:500; ab115998) and β-actin (1:500; ab8227) (all from Abcam, Cambridge, MA, USA) at at 4°C overnight. The blots were then washed with 0.1% Tween-PBS and incubated with goat anti-rabbit secondary antibodies (1:500; ab218695; Abcam) for antibody for 1 h at room temperature. The blots were then detected by enhanced chemiluminescence (ECL).
3
Western Blot Analysis of Vascular Proteins
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Total proteins were extracted from VSMCs or the aorta tissues according to the manufacturer’s instructions. In brief, equal amounts of protein was separated by SDS-PAGE and transferred onto an activated PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA) after electroblotting. After blocking with 5% non-fat milk for 2 h, the membrane was incubated with the primary antibodies overnight at 4 °C. The detailed information of primary antibodies were as follow: GADPH (ab181602, Abcam, 1:1000), phosphorylated-β-catenin (ab75777, Abcam, 1:500), β-catenin (ab32572, Abcam, 1:5000), Wnt inhibitory factor-1(WIF) (ab155101, Abcam, 1:2000), Runx2 (ab23981, Abcam, 1:1000), phosphorylated-AKT (ab81283, Abcam, 1:5000), Protein Kinase B (AKT) (ab188099, Abcam, 1:2000), receptor activator of NF-kB Ligand (RANKL) (ab239607, Abcam, 1:1000) and alkaline phosphatase (ALP) (ab228636, Abcam, 1:1000), phosphorylated-STAT3 (ab76315, Abcam, 1:2000), STAT3 (ab109085, Abcam, 1:1000), BMP2 (ab214821, Abcam, 1:1000). The membrane was rinsed with TBST three times (5 min/once), and then incubated at RT for 2 h with secondary antibody. After incubation, the membrane was rinsed three times with TBST (5 min/once). By using an enhanced chemiluminescence kit, protein bands were visualized by GeneGnome chemiluminescence imaging system.
4
Western Blot Analysis of Signaling Proteins
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Tissues or cells were lysed with RIPA buffer, separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. The proteins were then immunoblotted with the primary antibody: anti-SGK1 (bms-33275 M, Bioss), anti-pSTAT3 (bs-16558R, Bioss), anti-STAT3 (ab109085, Abcam), anti-NRF2 (bs-1074R, Bioss), anti-ERK1 + ERK2 (ab184699, Abcam), anti-pERK1 + pERK2 (ab214036, Abcam), anti-β-actin (4970, Cell Signaling Technology), anti-MPO (ab208670, Abcam), anti-CitH3 (ab281584, Abcam), anti-NE (bs-6982R, Bioss), anti-PAD4 (ab214810, Bioss). This was followed by color development using horseradish peroxidase (HRP)-enhanced chemiluminescence (ECL) assays.
5
Western Blot Protein Quantification Protocol
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The cultured cells were lysed using RIPA buffer (Thermo Scientific, Rockford, IL, USA), and the protein was extracted according to the manufacturer’s instructions. Total proteins were determined using a Bicinchoninic Acid Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). The samples were loaded onto 10 % SDS-polyacrylamide gel electrophoresis separating gel and transferred onto polyvinylidene difluoride membranes (Roche Diagnostics, Indianapolis, IN, USA). Primary antibodies used were as follows: goat polyclonal anti-p-JAK2 (sc-21870; 1:500; Santa Cruz), rabbit polyclonal anti-JAK2 (SC-294; 1:500; Santa Cruz), rabbit monoclonal anti-STAT3 (ab109085; 1:1000; Abcam), rabbit monoclonal anti-p-STAT3 (ab76315; 1:1000; Abcam), mouse monoclonal anti-CNTFR (sc-393214; 1:500; santa cruz), rabbit polyclonal anti-GP130 (3732; 1:1000; Cell Signaling Technology, Beverly, MA, USA), rabbit monoclonal to GFAP (ab68428, 1:2000; Abcam), and rabbit monoclonal anti-GAPDH (ab181602; 1:2000; Abcam). Horseradish peroxidase-conjugated anti-mouse, anti-rabbit, and anti-goat secondary antibodies (1:1000; Santa Cruz Biotechnology) were used. Color development was achieved by using the ECL Western Blotting Detection Kit (Thermo Scientific). Quantification of protein expression level was performed using the ImageJ analysis software.
6
Western Blot Analysis of Protein Regulators
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Total proteins were prepared in the RIPA lysis buffer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted to separate the protein samples. Then, proteins were electroblotted onto polyvinylidene difluoride membranes, blocked, and incubated with antibodies against TRIM18 (Abcam, Cambridge, United Kindom; ab70770), connective tissue growth factor (CTGF; Abcam; ab209780), α-SMA (Cell Signaling Technology, Danvers, MA, United States; #19245), STAT3 (Abcam; ab109085), p-STAT3 (Abcam; ab76315), protein tyrosine phosphatase non-receptor type 6 (SHP-1; Abcam; ab32559), suppressor of cytokine signaling 1 (SOCS1; Abcam; ab62584), PTP1B (Abcam; ab244207), protein inhibitor of activated STAT 1 (PIAS1; Abcam; ab109388), and GAPDH (CST; #5174) at 4°C for 12 h, followed by secondary antibodies (Beyotime). Bands were determined using the Quantity One software.
7
Protein Expression Analysis in Cells
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Tissues or cell lysates were isolated using cell lysis buffer (Beyotime, Nanjing, China). The membrane was transferred and blocked at 37°C for 1 h, followed by primary antibodies incubation, rabbit anti-MCM7 (1:1000, ab52489), PCNA (1:2000, ab92552) and Ki-67 (1:1000, ab16667), Bax (1:5000; ab32503), Bcl-2 (1:1000, ab59348), Cleaved caspase-3 (1:1000, ab49822), Cleaved caspase-9 (1:1000, ab2324), JAK2 (1:5000, ab108596), p- JAK2 (1:5000, ab32101), STAT3 (1:2000, ab109085), p-STAT3 (1:2000, ab76315) and rabbit anti-β-actin (1:2000, ab8227) were purchased from Abcam (Cambridge, UK), and then re-probed with immunoglobulin G (IgG) complexed to horseradish peroxidase (1:2000, ab6721) antibody. The chemiluminescence kit (Millipore, Germany) was adopted to observe the immune response zone and Image J software (ImageJ Software Inc., USA) was used to quantify the integrated density of each band. Antibodies mentioned before were supplied by Abcam (Cambridge, UK).
8
Quantitative Protein Analysis of Ovarian Cancer Cells
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Total protein from ovarian cancer cells under various treatments was extracted with Radioimmunoprecipitation lysis buffer. Then, the prepared protein concentration was determined using a Bicinchoninic Acid (BCA) Protein Kit (Beyotime; #P0012S). Subsequently, proteins were separated by 10% Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (ThermoFisher). Then, the membranes were blocked with 5% skimmed milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST). After rinsing, the membranes were incubated overnight at 4 °C with the primary antibodies against human ANGPTL3 (1:2000; #ab118208), JAK2 (1:5000; #ab108596), p-JAK2 (1:6000; #ab32101), STAT3 (1:1500; #ab109085) and p-STAT3 (1:1000; #ab267373) (all from Abcam, Cambridge, UK, USA). After washing with TBST three times, horseradish peroxidase-conjugated secondary antibody was added at room temperature for 2 h. Then, the binding bands were detected using the enhanced chemiluminescence (ECL) reagent (Beyotime) and visualized using a Gel DocTM XR imaging system (Bio-Rad Laboratories, Hercules, CA, USA). All bands were quantified using an Image J software (National Institutes of Health; Bethesda, MD, USA).
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