Ab156870

Manufactured by Abcam

Sourced in United States

Ab156870 is a laboratory equipment product. It is designed for general laboratory use.

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Lab products found in correlation

9 protocols using ab156870

Circinteractome-based Luciferase Assay for miRNA-circ_0038467 Interaction

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Bioinformatic analysis for the directly targeted miRNAs of circ_0038467 was carried out using the online database Circinteractome (https://circinteractome.nia.nih.gov/miRNA_Target_Sites/mirna_target_sites.html). The spartial sequences of circ_0038467 harboring wild‐type miR‐338‐3p‐binding sites (UGCUGG) and the site‐directed mutant (ACGACC) in the target region were cloned in the pmirGLO vector (Promega, Southampton, UK) downstream from the luciferase reporter gene, respectively. Each reporter construct (20 ng) was transfected into 16HBE cells together with miR‐338‐3p mimic (30 nM) or miR‐NC mimic (30 nM) using the HiPerFect transfection reagent. Cell extracts were prepared 48 hours post‐transfection, and the luciferase activity was determined by the Promega Dual‐luciferase Reporter Assay System.
For RIP studies, cell extracts were incubated with antibodies against Argonaute2 (ab156870, anti‐Ago2, Abcam) or a negative IgG (ab205718, anti‐IgG, Abcam) at 4°C for two hours, followed by the incubated with protein A/G beads (Sigma‐Aldrich) for two hours. Total RNA was obtained from the beads, and circ_0038467 enrichment was assessed by qRT‐PCR.

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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Western Blot Analysis of Protein Expression

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Total protein isolation was conducted in cells or tissues utilizing RIPA buffer (C0481, Sigma‐Aldrich), with the concentration estimated using the BCA kit (23227). Protein was uploaded onto SDS‐PAGE and electroblotted to PVDF membranes that received 1‐h blocking using BSA (5%) at ambient temperature. Membranes received overnight probing with primary antibodies at 4°C before 1.5‐h re‐probinh with HRP‐tagged goat anti‐rabbit IgG (1:20000, ab205718, Abcam) or goat anti‐mouse IgG (1:20,000, ab197767, Abcam) at room temperature. Next, developing solution (NCI4106, Pierce Biotechnology Inc., Rockford, IL, USA) was added to membranes, and band intensities were quantified using ImageLab software (Bio‐Rad Laboratories). The ratio of the gray value of the target band to that of internal reference protein GAPDH was representative of the relative protein expression of target genes. In this assay, the used primary antibodies consisted of antibody from Santa Cruz Biotechnology, Inc. to CXCL10 (mouse, 1:500, sc‐374092), and antibodies from Abcam to collagen I (rabbit, 1:1000, ab34710), collagen III (rabbit, 1:2000, ab7778), NOX2 (rabbit, 1:5000, ab129068), NOX4 (rabbit, 1:1000, ab154244), AGO2 (rabbit, 1:1000, ab156870), apolipoprotein B‐100 (apoB‐100) (1:1000, ab231574), and GAPDH (rabbit, 1:5000, ab194486).

Chen Y., Chen X., Li H., Li Y., Cheng D., Tang Y, & Sang H. (2021). Serum extracellular vesicles containing MIAT induces atrial fibrosis, inflammation and oxidative stress to promote atrial remodeling and atrial fibrillation via blockade of miR‐485‐5p‐mediated CXCL10 inhibition. Clinical and Translational Medicine, 11(8), e482.

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Profiling miR-502-3p and PRKAG2-AS1 Binding

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The relationships between PRKAG2-AS1, miR-502-3p and Ago2 were detected using RIP kits (Millipore, USA). The HCCLM3 and PLC5 cells were lysed with RIPA lysis buffer lasting 5 min. After being resuspended with RIP wash buffer, beads were incubated with 5 μg antibodies against Ago2 (1:50, ab156870; Abcam) and IgG (1:100, ab133470; Abcam) for binding. Then, following being resuspended, the magnetic bead-antibody complexes were harvested on a magnetic base. The samples and inputs were digested using proteinase K. Extracted RNA was detected using RT-qPCR.

Ou Y., Deng Y., Wang H., Zhang Q., Luo H, & Hu P. (2021). Targeting Antisense lncRNA PRKAG2-AS1, as a Therapeutic Target, Suppresses Malignant Behaviors of Hepatocellular Carcinoma Cells. Frontiers in Medicine, 8, 649279.

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Functional validation of circ_0000144-miR-623-GPRC5A axis

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The segmental sequence of circ_0000144 harboring the miR-623-pairing sites, the 3′-untranslated region (3′-UTR) of GPRC5A and their mutants in the seed sites were inserted by restriction endonuclease into the pmirGLO vector (Promega, Charbonnières, France), respectively, as described previously [20 (link)]. These constructs were transfected into HGC-27 and AGS cells, respectively, together with miR-623 mimic or miRNA NC mimic and tested for luciferase activity using the Promega Dual-luciferase Reporter Assay Kit. Renilla and Firefly luciferase activities were tested 48 h post-transfection using a luminometer (Berthold, Thoiry, France) based on the guidance of producers.
In RIP assays, the preparation of cell lysates was performed using the Complete Lysis-M reagent (Roche, Sussex, U.K.). After that, cell lysates were incubated with antibody against Argonaute2 (anti-Ago2, ab156870, Abcam, Cambridge, U.K.) or IgG (anti-IgG, ab150077, Abcam) for 2 h at 4°C before adding the protein A/G agarose (Sigma-Aldrich, Sydney, NSW, Australia) for 2 h. Beads were harvested, and total RNA was isolated, followed by the detection of the levels of circ_0000144, miR-623 and GPRC5A.

Mi L., Lei L., Yin X., Li N., Shi J., Han X., Duan X., Zhao M., Han G., Wang J, & Yin F. (2020). Circ_0000144 functions as a miR-623 sponge to enhance gastric cancer progression via up-regulating GPRC5A. Bioscience Reports, 40(8), BSR20201313.

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Detecting miR-141-3p and SIRT1 Expression

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RIP kits (0101, Guangzhou Geneseed Biotech Co., Ltd., Guangzhou, China) were used to detect the binding of miR-141-3p to SIRT1 3’UTR. Cells were lysed with an equal volume of RIPA lysate (P0013B, Beyotime) for 5 min, and centrifuged at 14000 rpm for 10 min at 4° C to take the supernatant. Part of the cell extract was taken as input, and part of it was incubated with rabbit anti-AGO2 (1:100, ab156870, Abcam) at room temperature for 30 mi, with rabbit anti-human IgG (1:100, ab109489, Abcam) as an NC. The sample was digested with proteinase K to extract RNA, which was used for subsequent RT-qPCR to detect miR-141-3p and SIRT1 expression.

Zhan Y., Li L., Guo C., Zhang Y., Zhao L., Tao Z., Zhang H, & Chen S. (2024). MicroRNA-141-3p reduces pulmonary hypoxia/reoxygenation injury through suppression of Beclin-1-dependent autophagy. Aging (Albany NY), 16(2), 1352-1373.

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Immunoprecipitation of RNA-binding Proteins

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AGS and HGC27 cells were lysed with the cell lysis buffer in Magna RIP® RNA‐Binding Protein Immunoprecipitation Kit (17‐700, Millipore, Temecula, CA, USA) following specific instructions. The lysates were then incubated with RIP buffer containing magnetic beads conjugated with human anti‐Ago2 antibody (ab156870, Abcam) or nonspecific IgG antibody (ab37450, Abcam) for 18 h under 4°C. TRIzol (15596026, Invitrogen, Shanghai, China) was used to extract the immunoprecipitated RNAs, followed by qRT‐PCR detection.

Wang Y., Zou R., Li D., Gao X, & Lu X. (2024). Exosomal circSTRBP from cancer cells facilitates gastric cancer progression via regulating miR‐1294/miR‐593‐3p/E2F2 axis. Journal of Cellular and Molecular Medicine, 28(8), e18217.

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Profiling miR-153-5p binding to TLR4 mRNA

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We collected Neuro-2a cells with transfection (TLR4 + miR-153-5p mimic or TLR4 + mimic control), and the Neuro-2a cells were lysed with Cell lysis buffer in the RIP kit (KT100-01, GZSCBio, China). Cells in the RNasefree EP tubes were labelled as the IgG + mimic-NC group, anti-Argonaute-2 (Ago2) + mimic-NC group, IgG + miR-153-5p group, Ago2 + miR-153-5p group, Inputmimic-NC group, and Input-miR-153-5p group, respectively. The antibodies used were the Ago2 antibody (1 : 50, ab156870, Abcam, USA) and the NC antibody IgG provided in the kit. In RNA binding protein immunoprecipitation, the magnetic bead-antibody mixture was mixed with cell lysate for incubation overnight (at 4°C). The Input-mimic-NC group and the Input-miR-153-5p group were not given the magnetic bead-antibody mixture. On the second day, the purified RNA was analysed for the expression of TLR4 by qRT-PCR, and the enrichment level was calculated.

Zha H., Miao W., Rong W., Wang A., Jiang W., Liu R., Liu L, & Wang Y. (2021). Remote ischaemic perconditioning reduces the infarct volume and improves the neurological function of acute ischaemic stroke partially through the miR-153-5p/TLR4/p65/IkBa signalling pathway. Folia neuropathologica, 59(4).

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AGO2-Mediated RIP Protocol

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RIP was performed with an EZ-Magna RIP Kit (Millipore, USA) according to the product manual. The AGO2 antibody was used for RIP. In brief, 1 × 10⁶ cells were lysed in 1 ml lysis buffer containing 1% protease inhibitor and RNase inhibitor on ice for 10 min. These cell lysates were centrifuged at 12000 g for 20 min at 4°C, and the supernatant was collected. Then, 100 μl of supernatant was retained as input control. Following this, 500 μl of protein A+G beads were incubated with 5 μg of IgG or AGO2 antibody (Abcam, ab156870) and 450 μl supernatant at 4°C for 12 h on a shaking table at 15 rpm/min. The coimmunoprecipitated RNAs were detected by qRT-PCR.

Yu J., Zhang X., He R., Yang X., Hu J., Tan F., Yao J., Lei X, & Wen G. (2022). LINC01234 Accelerates the Progression of Breast Cancer via the miR-525-5p/Cold Shock Domain-Containing E1 Axis. Disease Markers, 2022, 6899777.

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Comparative Protein Expression Analysis

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Protein samples were extracted from cells by using RIPA buffer (Thermo Scienti c) supplemented with the protease inhibitor cocktail (MedChemExpress, NJ, USA). Total protein concentrations were quantitated via the BCA method (Thermo Scienti c), and then equal amounts of proteins samples were subjected to SDS-PAGE, followed by immunoblotting. Primary antibodies anti-ADH1A (ab108203), anti-ADH1B (ab175515), anti-ADH1C (ab168748), anti-ADH4 (ab137077), anti-ADH5 (ab177932), anti-AGO1 (ab5070), anti-AGO2 (ab156870) were purchased from Abcam (Cambridge, MA, USA). Anti-ADH6 was purchased from Santa Cruz (CA, USA), and anti-β-Actin (AC-15, Boster, Wuhan, China) was used as a control antibody. After HRP-conjugated secondary antibody incubation, the target band was detected using the chemiluminescence system (Tanon 5200, Shanghai.China).

Chen N., Luo J., Hou Y., Ji Y., Xie M., Ge S., & Yu D. (2022). miR-29c promotes alcohol dehydrogenase gene cluster expression by activating the enhancer within ADH6.

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