Ab138005

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab138005 is a lab equipment product from Abcam. It is a tool designed to assist in scientific research and experimentation.

Automatically generated - may contain errors

Lab products found in correlation

Ab133666, by Abcam (11 mentions) Ab68153, by Abcam (6 mentions) Ab76315, by Abcam (6 mentions) Ab267373, by Abcam (4 mentions) Ab119352, by Abcam (4 mentions) Ab9485, by Abcam (4 mentions) Ab8245, by Abcam (3 mentions) Ab47435, by Abcam (3 mentions) Ab181602, by Abcam (3 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions) Ab32503, by Abcam (2 mentions) Ab30647, by Abcam (2 mentions) Ab76055, by Abcam (2 mentions) Ab76003, by Abcam (2 mentions) Bca kit, by Beyotime (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Ripa lysis solution, by Beyotime (2 mentions) Ab16030, by Abcam (2 mentions) Ab29045, by Abcam (2 mentions) Ab47425, by Abcam (2 mentions)

20 protocols using ab138005

Immunoblotting Analysis of Signaling Proteins

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The treated cells were washed twice with cold 1X phosphate-buffered saline, lysed with RIPA lysate buffer for 20 min, and centrifuged (12,000 rpm, 10 min, 4°C) to obtain the supernatant. The loading buffer was added, and the supernatant was denatured at 95°C for 5 min until protein denaturation was achieved. Electrophoresis analysis of protein samples (10 μL/well) was performed using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Proteins were transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). The blots were blocked with 5% nonfat milk powder for 1 h and incubated with corresponding primary antibody at 4°C overnight. On the second day, the blots were cleaned with 1X tris-buffered saline and incubated with the appropriate secondary antibodies at room temperature for 1 h. Protein brands were detected by chemiluminescence (Millipore, Burlington, USA), and the expression level was determined using Image J.
Primary antibodies against JAK (ab133666), anti-p-JAK (ab138005), anti-STAT1 (ab109320), anti-p-STAT1 (ab109461), mTOR (ab32028), p-mTOR (ab109268), p-AKT (ab105731), Bax (ab32503), and Bcl2 (ab182858) were purchased from Abcam (Cambridge, UK). The following secondary antibodies were purchased from GenScript (Piscataway, NJ, USA): goat anti-rabbit IgG (H&L) and goat anti-mouse IgG (H&L).

Guo L., Sun J., Wang C., Wang Y., Wang Y., Li D, & Li Y. (2022). Epirubicin Enhances the Anti-Cancer Effects of Radioactive 125I Seeds in Hepatocellular Carcinoma via Downregulation of the JAK/STAT1 Pathway. Frontiers in Oncology, 12, 854023.

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Western Blot Analysis of Signaling Proteins

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Proteins were extracted from experimental cells in the RIPA lysis buffer (Invitrogen, USA) by 10-minute centrifugation at 18,000 × g at 4° C. Proteins (20 μg/lane) were separated by using SDS-PAGE (10% to 15%) and were then transferred onto the PVDF membranes, which were blocked by 5% (w/v) milk mixed in Tris-buffered saline (TBS) with 0.2% (v/v) Tween-20 (TBST) for 2 hours at room temperature (RT). Membranes with proteins were incubated with the indicated primary antibodies: CFHR3 (16583-1-AP, Proteintech, USA), JAK1 (ab133666, Abcam, UK), p-JAK1 (ab138005, Abcam, UK), STAT3 (ab68153, Abcam, UK), p-STAT3 (ab267373, Abcam, UK), p53 (ab26, Abcam, UK) and β-actin (sc-81178, Santa Cruz, USA) at 4° C overnight. After that, membranes were washed with 1× TBST three times and incubated with corresponding secondary antibodies at RT for 1.5 hours. The protein bands then were visualized and analyzed using an ECL system (Amersham, USA).

Wan Z., Li X., Luo X., Wang B., Zhou X, & Chen A. (2022). The miR-590-3p/CFHR3/STAT3 signaling pathway promotes cell proliferation and metastasis in hepatocellular carcinoma. Aging (Albany NY), 14(14), 5783-5799.

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Western Blot Analysis of Jak1/Stat3 Signaling

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Protein samples used for western blotting were extracted using RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors (Roche, China). The protein samples were quantified by the BCA™ Protein Assay Kit (Beyotime Biotechnology). Then, equal amounts of samples were separated by 10–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were incubated overnight at 4°C with primary antibodies p-Jak1 (1:500; ab138005; Abcam Biotechnology, USA), p-Stat3 (1:500; ab30647; Abcam), and GAPDH (1:1000; ab8245; Abcam). Then, membranes were washed and incubated with horseradish peroxidase-marked secondary antibodies for 1 h at room temperature. After that, membranes were transferred into the Bio-Rad ChemiDoc™ XRS system (Bio-Rad Laboratories, USA) and 200 μL of Immobilon Western Chemiluminescent HRP Substrate (Millipore) was added to cover the membrane surface. Protein signals were captured and the intensities of the bands were quantified with the Bio-Rad Image™ 3.0 version software (Bio-Rad).

Gao F., Tan Y, & Luo H. (2020). MALAT1 is involved in type I IFNs-mediated systemic lupus erythematosus by up-regulating OAS2, OAS3, and OASL. Brazilian Journal of Medical and Biological Research, 53(5), e9292.

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Inhibition of JAK/STAT Signaling in NSCLC

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H1975 or HCC827GR cells (0.8×10⁶) were seeded in a 60-mm plate and allowed to adhere overnight. The cells were then treated with gefitinib, or ZA, or gefitinib + ZA, or vehicle for 48 h. Cells were lysed by RIPA lysis buffer, and 30 µg protein was resolved by 10% sodium dodecyl sulphate-polyacrylamide gel, following which the protein bands were transferred to nitrocellulose membranes. The membrane was blocked with 5% skim milk and probed with primary antibodies with gentle shaking at 4°C overnight. The membranes were then washed three times with TBST, and then incubated with the appropriated secondary antibodies for 2 h. Antibody-bound proteins were detected by ECL Select Western Blotting Detection Reagent (GE Healthcare). The following primary antibodies were used: anti-JAK1 (ab133666, Abcam), anti-phosphorylated (p)-JAK (ab138005, Abcam), anti-STAT3 (ab119352, Abcam), anti-p-STAT3 (ab76315, Abcam), anti-E-cadherin (ab76055, Abcam), anti-vimentin (ab8978, Abcam), and anti-SNAIL (ab180714, Abcam). ImageJ software v1.43 (National Institutes of Health, Bethesda, MD, USA) was used to quantify the density and size of the blots.

Yang X., Gao Y., Liu Q., Wan L., Liu H., Bian W., Du Y, & Huang C. (2020). Zoledronic acid re-sensitises gefitinib-resistant lung cancer cells by inhibiting the JAK/STAT3 signalling pathway and reversing epithelial-mesenchymal transition. Oncology Reports, 45(2), 459-468.

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Immunoblotting for ERBB2, JAK-STAT Pathway

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The RIPA extraction reagent (Beyotime, Shanghai, China) were used to extract proteins. The protein was then separated from the sample buffer using SDS‐PAGE, transferred to PVDF membranes, and blocked for 1 h with 5% skim milk. Primary antibodies were provided from Abcam company, anti‐ERBB2 (1:1000, ab134182), anti‐JAK1 (1:1000, ab138005), anti‐JAK2 (1:1000, ab267373), anti‐STAT3 (1:1000, ab68153), anti‐STAT5 (1:1000, ab32364), anti‐GAPDH (1:1000, ab8245). Then, bands were visualized by a chemiluminescence (ECL) kit (Boster, China) and analysed by ImageJ.

Li L., Zhang Y., Yang K., Liu W., Zhou Z, & Xu Y. (2024). miRNA‐449c‐5p regulates the JAK‐STAT pathway in inhibiting cell proliferation and invasion in human breast cancer cells by targeting ERBB2. Cancer Reports, 7(2), e1974.

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Investigating JAK/STAT Signaling in TSLP-Induced Immune Responses

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Antibodies for p-JAK1 (ab138005), p-JAK2 (ab32101), and TSLP (ab188766) were purchased from Abcam (Cambridge, UK). β-Actin (AF5001) antibody and carboxyfluorescein diacetate succinimidyl ester (CFSE, C1031) were purchased from Beyotime (Shanghai, China). JAK1 (66466-1-Ig), JAK2 (17670-1-AP), and STAT3 (10144-2-AP) were obtained from Proteintech (Wuhan, China). p-STAT3 (9145T) antibody was purchased from CST (Danvers, MA). Antibody for RORγt (14-6988-82) was purchased from Thermo Fisher. TSLPR (SAB2900760) antibody was obtained from Sigma-Aldrich. Antibodies for APC-CD3 (100236), FITC-CD4 (100406), APC-IL-17A (506916), APC-MHC-II (107614), APC-CD80 (104714), APC-CD86 (105012), and FITC-CD11c (117306) were purchased from BioLegend (San Diego, CA). Recombinant mouse TSLP (rmTSLP) (555-TS-010) was derived from R&D System (Minneapolis, MN). Ruxolitinib (S1378) and BBI608 (S7977) were purchased from Selleck Chemicals (Houston, TX).

Han F., Guo H., Wang L., Zhang Y., Sun L., Dai C, & Wu X. (2020). TSLP Produced by Aspergillus fumigatus-Stimulated DCs Promotes a Th17 Response Through the JAK/STAT Signaling Pathway in Fungal Keratitis. Investigative Ophthalmology & Visual Science, 61(14), 24.

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Protein Quantification and Western Blot Analysis

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The proteins were extracted to determine the concentration as per the instructions of a bicinchoninic acid kit (Thermo Scientific Pierce, Rockford, IL, USA). The extracted proteins were boiled and run on sodium dodecyl sulfate polyacrylamide gel electrophoresis from 80 to 120 v. Afterwards, the proteins were transferred into the polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked and incubated with primary antibodies at 4°C overnight: SOCS5 (1:1000, ab97283, Abcam), Janus kinase 1 (JAK1, 1:1000, ab47435, Abcam), p-JAK1 (1:1000, ab138005, Abcam), signal transducer and activator of transcription 3 (STAT3, 1:5000, ab119352, Abcam) and p-STAT3 (1:2000, ab76315, Abcam). Then the membrane was rinsed in tris-buffered saline tween (TBST), and cultured for 1 h with secondary antibody (ZSGB-BIO, Beijing, China) labeled by horseradish peroxidase. After TBST washing, the proteins were visualized by enhanced chemiluminescence reagent and developed by Gel EZ imager (Bio-Rad Laboratories, CA, USA). Finally, the target bands were analyzed with Image J software (National Institutes of Health, Bethesda, Maryland, USA) for gray value analysis.

Wu L., Xia J., Li D., Kang Y., Fang W, & Huang P. (2020). Mechanisms of M2 Macrophage-Derived Exosomal Long Non-coding RNA PVT1 in Regulating Th17 Cell Response in Experimental Autoimmune Encephalomyelitisa. Frontiers in Immunology, 11, 1934.

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Western Blot Analysis of Inflammatory Cytokines

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Cell extracts were prepared using the Complete Lysis‐M reagent (Roche Molecular Systems, CA, USA), and western blot was performed as previously described.12 Protein samples (50 μg) were resolved on 8%–10% sodium dodecyl sulfate polyacrylamide gel (SDS‐PAGE), electrophoretically blotted onto a Hybond P polyvinylidene fluoride membrane (PVDF, Millipore, Vienna, Austria), and then probed with primary antibodies: anti‐IL‐6 (ab6672), anti‐IL‐8 (ab7747), anti‐IL‐1β (ab9722), antiphosphorylated JAK (anti‐p‐JAK, ab138005), anti‐JAK (ab133666), anti‐STAT3 (ab119352), anti‐p‐STAT3 (ab76315) and anti‐glyceraldehyde 3‐phosphate dehydrogenase (anti‐GAPDH, ab181602, Abcam, Cambridge, UK).

Liu G., Wan Q., Li J., Hu X., Gu X, & Xu S. (2020). Circ_0038467 regulates lipopolysaccharide‐induced inflammatory injury in human bronchial epithelial cells through sponging miR‐338‐3p. Thoracic Cancer, 11(5), 1297-1308.

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Protein Expression Analysis via Western Blot

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RIPA lysis buffer was used to extract the total proteins from the cells, and the protein concentration was quantified using a BCA kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. A total of 30 µg protein/lane was loaded on a 10% SDS-gel, resolved using SDS-PAGE and transferred to a PVDF (Roche Diagnostics GmbH). The membranes were blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with the corresponding primary antibodies anti-NCAPG (1:800, ab70350, Abcam), anti-Bcl-2 (1:1,000, ab32124, Abcam), anti-Bax (1:1,000, ab182733, Abcam), anti-Bad (1:1,000, ab32445, Abcam), anti-MMP2 (1:1,000, ab92536, Abcam), anti-MMP9 (1:1,000, ab76003, Abcam), anti-p-EGFG (1:1,000, ab40815, Abcam), anti-EGFR (1:1,000, ab52894 Abcam), anti-p-JAK1 (1:1,000, ab138005, Abcam), anti-JAK1 (1:800, ab133666, Abcam), anti-STAT3 (1:1,000, ab68153, Abcam), anti-p-STAT3 (1:1,000, ab76315, Abcam), anti-GAPDH (1:1,000, ab9485, Abcam) overnight at 4˚C. The following day, the membranes were incubated with goat anti-rabbit IgG H&L (HRP)-conjugated secondary antibodies (1:5,000, ab7090, Abcam) for 1 h at room temperature. The expression levels of the different proteins were detected using enhanced chemiluminescence reagent (Bio-Rad Laboratories, Inc.). The data were analyzed using ImageJ version 1.46 (National Institutes of Health).

Li J., Zheng J., Lin B., Sun H., Lu S., Wang D, & Huo H. (2023). Knockdown of NCAPG promotes the apoptosis and inhibits the invasion and migration of triple‑negative breast cancer MDA‑MB‑231 cells via regulation of EGFR/JAK/STAT3 signaling. Experimental and Therapeutic Medicine, 25(3), 119.

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Western Blot Analysis of Extracellular Vesicle Markers

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Cell proteins were extracted with RIPA lysis buffer (Thermo Fisher, USA). The protein was extracted by incubation, vortex, and centrifugation (15,000 ×g, 4°C for 25 min). A BCA reagent kit (Beyotime, China) was used to measure the protein concentrations. Total protein was separated by SDS-PAGE gel (100 V, 1.5 h) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA) (50 V, 80 min). After blocking in 5% nonfat milk for 1 hour, the membranes were incubated overnight at 4°C with the indicated primary antibodies, including anti-CD63 (Abcam, ab68418), anti-TSG101 (Abcam, ab133586), anti-E-cadherin (Abcam, ab76055), anti-N-cadherin (Abcam, ab76011), anti-vimentin (Abcam, ab20346), anti-JAK1 (Abcam, ab138005), anti-STAT3 (Abcam, ab68153), IL-10 (Abcam, ab215975), arginase-1 (ab133543) and anti-GAPDH (Abcam, ab9485). They were then incubated with secondary antibodies for 1 hour at room temperature and visualized by the ECL chemiluminescence reagent (Millipore, USA).

Huang X., Wang J., Guan J., Zheng Z., Hao J., Sheng Z., Wang M., Xu T., Guo G, & Yao L. (2022). Exosomal Circsafb2 Reshaping Tumor Environment to Promote Renal Cell Carcinoma Progression by Mediating M2 Macrophage Polarization. Frontiers in Oncology, 12, 808888.

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