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Ab31645

Manufactured by Abcam
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Ab31645 is a laboratory equipment product offered by Abcam. It serves as a core functional component for research applications. Further details about its intended use or specifications are not available.

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Lab products found in correlation

Ab209795, by Abcam (2 mentions) Pa5 29813, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Ab79852, by Abcam (1 mentions) Ab92726, by Abcam (1 mentions) Total protein extraction kit, by Solarbio (1 mentions) Sa00001 1, by Proteintech (1 mentions) Sa00001 2, by Proteintech (1 mentions) G8795, by Merck Group (1 mentions) Ab114126, by Abcam (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Anti myd88, by Cell Signaling Technology (1 mentions) Ab51324, by Abcam (1 mentions) Anti irak4, by Cell Signaling Technology (1 mentions) Apoptosis antibody sampler kit, by Cell Signaling Technology (1 mentions) Ab9332, by Abcam (1 mentions) Sc 20680, by Santa Cruz Biotechnology (1 mentions) 1mod 1a9, by Euromedex (1 mentions) Bis tris sds gels, by Bio-Rad (1 mentions) Ab32127, by Abcam (1 mentions)

13 protocols using ab31645

1

Western Blot Protein Analysis

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The total proteins were prepared, and their concentrations were detected using a Total Protein Extraction Kit (Solarbio, Beijing, China). Cell lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, USA). Then, 5% non-fat milk in TBST buffer was used for the blockage of membranes containing protein. The primary antibodies used in this study were: anti-HSP70 (1: 1000, Abcam, ab79852), anti-hnRNPA2B1 (1: 1000, Abcam, ab31645), and anti-CD9 (1: 1000, Abcam, ab92726). HRP-conjugated secondary goat anti-mouse (1: 5000, Proteintech, Rosemont, IL, SA00001-1) or goat anti-rabbit (1: 5000, Proteintech, SA00001-2) antibodies were incubated for 2 h at room temperature. The relative grey values of immunoreactive bands were calculated based on GAPDH.
Zheng Z., Chen M., Xing P., Yan X, & Xie B. (2019). Increased Expression of Exosomal AGAP2-AS1 (AGAP2 Antisense RNA 1) In Breast Cancer Cells Inhibits Trastuzumab-Induced Cell Cytotoxicity. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 2211-2220.
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2

Developmental Chd8 and Hnrnpa2b1 Expression

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Isolated forebrain from e14.5 embryos (male and female littermates), P0 neonates, and P60 adults were lysed in 50 mM Tris HCl pH 8, 140 nM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40 and 0.25% Triton with protease inhibitor cocktail (Roche). After sonication, samples were spun down and the supernatant was used for a BCA Bradford assay using the Spectramax 190 plate reader to assess protein concentration using a standard curve. We ran 22 μg of protein on a 3-8% Tris Acetate gel using the Novex western blotting system (Invitrogen). Anti-Chd812 (link) (1:1,000, ab114126; Abcam), anti-Hnrnpa2b140 (link) (1:1,000, ab31645; Abcam), and anti-Gapdh41 (link) (1:10,000, G8795; Sigma-Aldrich) primary antibodies were incubated overnight in Fluorescent Blocker solution (Millipore), visualized using a LI-COR Odyssey CLx system and quantified in FIJI (National Institutes of Health). For Chd8 western blots, both males and females were used. For the Hnrnpa2b1 western blot, only male samples were used. Protein levels assayed via western blot were compared via Student’s t-test and one-way ANOVA for adult Chd8 levels.
Gompers A.L., Su-Feher L., Ellegood J., Copping N.A., Riyadh M.A., Stradleigh T.W., Pride M.C., Schaffler M.D., Wade A.A., Catta-Preta R., Zdilar I., Louis S., Kaushik G., Mannion B.J., Plajzer-Frick I., Afzal V., Visel A., Pennacchio L.A., Dickel D.E., Lerch J.P., Crawley J.N., Zarbalis K.S., Silverman J.L, & Nord A.S. (2017). Germline Chd8 haploinsufficiency alters brain development in mouse. Nature neuroscience, 20(8), 1062-1073.
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3

Protein Expression Analysis Protocol

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Anti-human CHD1L (ab51324), CHD1L (ab197019), anti-MYLK (ab232949), anti-Cyclin D1 (ab134175), and anti-hnRNP A2/B1 (ab31645) antibodies were purchased from Abcam. Anti-human GAPDH (#5174), anti-NF-κB pathway sampler kit (#9936), Apoptosis Antibody Sampler Kit (#9930), anti–rabbit IgG (#6990), anti–MLC2 sampler kit (#9776), anti-MyD88 (#4283), and anti-IRAK4 (#4363) Proteins were purchased from Cell Signaling Technology (CST).
Wang G., Zhang X., Cheng W., Mo Y., Chen J., Cao Z., Chen X., Cui H., Liu S., Huang L., Liu M., Ma L, & Ma N.F. (2021). CHD1L prevents lipopolysaccharide-induced hepatocellular carcinomar cell death by activating hnRNP A2/B1-nmMYLK axis. Cell Death & Disease, 12(10), 891.
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4

Subcellular Protein Fractionation and Detection

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Protein fractionation in the chromatin-bound, the nucleoplasmic or the cytoplasmic compartments was performed essentially as described [81 (link)]. The protein extraction was performed on 4 × 107 primary MEFs (passage 3) or on 4 × 107 R1 mouse ESCs. The protein fractions were separated on 4–20 % gradient Bis-Tris SDS gels (BioRad), blotted, and incubated with the following primary antibodies: HP1β (1MOD-1A9, Euromedex; 1:2000), HP1γ (2MOD-1G6, Euromedex; 1:2000), H3K9me3 (rabbit polyclonal; 1:100), kindly provided by T. Jenuwein (Freiburg), histone H3, kindly provided by M. Bustin (1:10,000, rabbit), and alpha tubulin (#ACLX135B, Accurate Chemical & Scientific Corporation). Other antibodies used for western blots included lamin A/C (sc-20680, SantaCruz; 1:100), hnRNPa2/b1 (ab31645, Abcam; 1:200), phosphoserine (ab9332, Abcam; 1:100) and phosphothreonine (Cell Signaling #93865; 1:3000).
Mattout A., Aaronson Y., Sailaja B.S., Raghu Ram E.V., Harikumar A., Mallm J.P., Sim K.H., Nissim-Rafinia M., Supper E., Singh P.B., Sze S.K., Gasser S.M., Rippe K, & Meshorer E. (2015). Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells. Genome Biology, 16, 213.
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5

Protein Expression Analysis in Prostate and Lung Cancer Cells

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LNCaP and NCI-H660 cells were harvested using radioimmunoprecipitation assay (RIPA) Lysis (Elabscience, Wuhan, China), and protein concentration were detected by the BCA protein assay kit (Pierce, Rockford, IL, USA). 10 µg of protein were separated by 10% SDS-PAGE gels, followed by electrotransfer onto PVDF membranes (Arkema, Paris, France). The membrane was incubated with primary antibody against ChgA (1:1000, ab254557, Abcam), NSE (1:100, ab105389, Abcam), SYP (1:1000, ab32127, Abcam), Hnrnpa2b1 (5 µg/ml, ab31645, Abcam), and β-actin (1:1000, 3700, CST, Danvers, MA, USA) overnight at 4 °C, HRP-conjugated secondary antibodies (1:1000; ab205718; Abcam), and visualized through the ECL imaging system (Bio-Rad, Hercules, CA, USA).
Liu B., Jiang H.Y., Yuan T., Luo J., Zhou W.D., Jiang Q.Q, & Wu D. (2021). Enzalutamide-Induced Upregulation of PCAT6 Promotes Prostate Cancer Neuroendocrine Differentiation by Regulating miR-326/HNRNPA2B1 Axis. Frontiers in Oncology, 11, 650054.
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6

Profiling Protein-RNA Interactions

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RIP assay was performed using Imprint RNA Immunoprecipitation Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, LUAD cell lysates were incubated with anti-Zeb1 antibody (ab276129, Abcam), anti-Siah1 (MA5-24781, Invitrogen), anti-HNRNPA2B1 (ab31645, Abcam), or the non-specific anti-IgG antibody (Abcam) at 4 °C overnight. Protein-RNA complexes were then captured using protein A magnetic bead and associated RNAs were analyzed by RT-qPCR.
Hu R., Xu B., Ma J., Li L., Zhang L., Wang L., Zhu J., Guo T., Zhang H, & Wang S. (2023). LINC00963 promotes the malignancy and metastasis of lung adenocarcinoma by stabilizing Zeb1 and exosomes-induced M2 macrophage polarization. Molecular Medicine, 29, 1.
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7

Western Blot Analysis of Endothelial Cell Proteins

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HUVECs were collected and lysed using a cell lysis solution containing complete protease inhibitors. Protein concentrations were determined using a BCA protein assay kit (Pierce, USA). Briefly, proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Hybond-P; GE Healthcare, Singapore). The membranes were incubated for 1 h in 5% nonfat milk and Tris-buffered saline with Tween-20, which was followed by an overnight incubation at 4°C with primary antibodies against the following: GAPDH (5174, Cell Signaling Technology), hnRNPA2/B1 (ab31645, Abcam), VE-cadherin (ab33168, Abcam), β-catenin (ab325728, Abcam), ICAM-1 (BBA3, R&D Systems), NF-κB p65 (ab16502, Abcam), and p-NF-κB p65 (ab86299, Abcam). The membranes were then incubated with goat anti-mouse IgG H&L (HRP) (ab6789, Abcam) or goat anti-rabbit IgG H&L (HRP) (ab6721, Abcam) for 2 h at room temperature. Proteins were visualized with an enhanced chemiluminescence kit (Pierce, USA) on a ChemiDocTM XRS+ system (Bio-Rad). The densities of the protein bands were quantified by ImageJ software using GAPDH as a reference.
Chen Y., Tang D., Zhu L., Yuan T., Jiao Y., Yan H, & Yu W. (2020). hnRNPA2/B1 Ameliorates LPS-Induced Endothelial Injury through NF-κB Pathway and VE-Cadherin/β-Catenin Signaling Modulation In Vitro. Mediators of Inflammation, 2020, 6458791.
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8

RNA Immunoprecipitation (RIP) Assay for Protein-RNA Interactions

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The RIP assay was performed using a RIP kit (Millipore, MA, USA) to verify the interaction between YWHAH and circEHD2, FUS with circEHD2 pre-mRNA, and hnRNPA2B1 with circEHD2. Briefly, approximately 2 × 107 OSRC-2 and 786-O cells were collected and lysed by RIP lysis buffer. The interaction RNA was precipitated by anti-YWHAH antibody (ab206292, Abcam), anti-FUS antibody (ab243880, Abcam), and anti-hnRNPA2B1 antibody (ab31645, Abcam). The anti-IgG antibody (ab172730, Abcam) was used as a negative control. Then, the coprecipitated RNAs were purified by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and analyzed by qRT–PCR. For the analysis of circEHD2 pre-mRNA, miR-200 was used as a positive control [26 (link)].
He T., Zhang Q., Xu P., Tao W., Lin F., Liu R., Li M., Duan X., Cai C., Gu D., Zeng G, & Liu Y. (2023). Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts. Molecular Cancer, 22, 117.
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9

Hnrnpa2b1 RNA-binding Protein Profiling

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C2C12 Hnrnpa2b1-WT and KO myoblasts were seeded at 6 × 106 cells per 15 cm plate, grown 24 hr at 37°C, 5% CO2, and harvested as undifferentiated myoblasts. Myoblasts were lysed in a CHAPS-based buffer and pre-cleared using protein-A bound Dynabeads (Thermo, 10001D). Pre-cleared cell lysates were incubated with rabbit polyclonal anti-Hnrnpa2b1 antibody (ab31645, Abcam). Antibody-bound Hnrnpa2b1 was bound to protein-A Dynabeads overnight and magnetically isolated from the whole-cell lysate. Hnrnpa2b1-bound RNA was isolated from the Dynabead-Antibody-Hnrnpa2b1 complex via TRIZol RNA purification. cDNA libraries were created from purified RNA via oligo-DT priming and SuperScriptIII-enzyme reverse transcription. cDNA libraries were probed against Hnrnpa2b1-eCLIP hits chosen as a subset of the significantly enriched Gene Ontology terms via quantitative, real-time PCR (qRT-PCR). Primers used target Gapdh, Hnrnpa2b1, Prpf19, Snrnp70, Sfpq, Mbnl1, Hnrnpa3, and Mef2a. (Primer sequence is given in Supplementary file 1.) qRT-PCR was performed using SYBR-Green qRT-PCR reagent (Bio-Rad), and fluorescent emission was measured using a Bio-Rad CFX384 Real-Time PCR Detection system.
Wheeler J.R., Whitney O.N., Vogler T.O., Nguyen E.D., Pawlikowski B., Lester E., Cutler A., Elston T., Dalla Betta N., Parker K.R., Yost K.E., Vogel H., Rando T.A., Chang H.Y., Johnson A.M., Parker R, & Olwin B.B. (2022). RNA-binding proteins direct myogenic cell fate decisions. eLife, 11, e75844.
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10

Protein Quantification and Detection

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Whole cell extracts were prepared in RIPA buffer containing a protease inhibitor cocktail. Total protein was separated by SDS-PAGE. The following antibodies were used: anti-hnRNPA2B1 (ab31645, Abcam, 1:500), anti-NEDD8 (ab81264, Abcam, 1:5000), anti-NF-κB1 (ab209795, Abcam, 1:1000), anti-TRAF2 (ab126758, Abcam, 1:1000), anti-MTPα (PA5-29813, Invitrogen, 1:1500), and anti-actin (ab8226, Abcam, 1:500).
Chen W., Wang Y., Xia W., Zhang J, & Zhao Y. (2022). Neddylation-mediated degradation of hnRNPA2B1 contributes to hypertriglyceridemia pancreatitis. Cell Death & Disease, 13(10), 863.
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