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Hur 3a2

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HuR (3A2) is a monoclonal antibody that specifically recognizes the HuR (Human antigen R) protein. HuR is an RNA-binding protein involved in the regulation of mRNA stability and translation. The 3A2 clone of this antibody can be used to detect and study the expression and localization of HuR in various experimental systems.

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Lab products found in correlation

Lamin a c, by Cell Signaling Technology (3 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Mouse pdl1 elisa kit, by Boster Bio (2 mentions) 10k protein concentrator pes, by Thermo Fisher Scientific (2 mentions) Hpdl1, by Cell Signaling Technology (2 mentions) Anti biotin hrp linked, by Cell Signaling Technology (2 mentions) Anti rabbit igg1 hrp linked antibodies, by Cell Signaling Technology (2 mentions) Goat anti mouse igg1 hrp antibodies, by Santa Cruz Biotechnology (2 mentions) Pd1 pdl1 inhibitor 1, by Cayman Chemical (2 mentions) Hif1a, by Cell Signaling Technology (2 mentions) Pd l1, by Santa Cruz Biotechnology (2 mentions) Cell lysis buffer, by Cell Signaling Technology (2 mentions) Donkey anti goat igg hrp, by Santa Cruz Biotechnology (2 mentions) Mln4924, by Active Motif (2 mentions) Mpdl1 b7 h1 antibody, by BD (2 mentions) Dc protein assay, by Bio-Rad (1 mentions) Ge10600001, by Merck Group (1 mentions) Cpeb4, by Abcam (1 mentions) Hif1a, by Cayman Chemical (1 mentions) Socs1, by Abcam (1 mentions)

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Anti-HuR (3A2, (5 citations) Anti-HuR antibody (3A2, (2 citations) HuR antibody (3A2, (2 citations)

6 protocols using hur 3a2

1

Western Blot Analysis of Protein Expression

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Protein extracts were quantified by DC Protein assay (Bio-Rad), and equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis. After transfer of the proteins onto a nitrocellulose membrane (GE10600001, Sigma) for 1 hr at 100 mV, membranes were blocked in 5% milk, and specific proteins were labeled with the following antibodies: CPEB4 (Abcam Ab83009/clone 149C/D10, monoclonal homemade); HuR (3A2, sc-5261 Santa Cruz); HIF1a (Cayman 10006421); phospho-p44/42 (Erk1/2) (Thr202/Try204) (Cell Signaling clone E10, 9106); SOCS1 (Abcam Ab9870); phospho-p38 (Thr180/Y182) (Cell Signaling, 9211S); p38α (C-20)-G (Santa Cruz, sc-535-G); phospho-MAPKAPK2 (Thr222) (Cell Signaling, 3044S), TTP (D1I3T) (Cell Signaling, 71632), and vinculin (Abcam Ab18058). Quantification was done with ImageStudioLite software, and protein expression was normalized by the loading control signal.
Suñer C., Sibilio A., Martín J., Castellazzi C.L., Reina O., Dotu I., Caballé A., Rivas E., Calderone V., Díez J., Nebreda A.R, & Méndez R. (2022). Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation. eLife, 11, e75873.
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2

Protein Expression Analysis by Western Blot

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Westerns were performed using the following antibodies α-Tubulin (B-5-1-2, 1:5000, Thermo Scientific); HuR (3A2, 1:4000, Santa Cruz Biotechnology) COX-2 (H-62, 1:1000, Santa Cruz), Total OXPHOS Human WB Antibody Cocktail (ab110411, 1:1000, Abcam), Anti-TOMM20 antibody (ab56783, 1:1000, Abcam), Beta-actin (13E5 1:10,000, Cell Signaling). Membranes were scanned using Odyssey Infrared Imaging System (LI-COR Biosciences) and analyzed with Image Studio Lite Version 5.2.
Schultz C.W., McCarthy G.A., Nerwal T., Nevler A., DuHadaway J.B., McCoy M.D., Jiang W., Brown S.Z., Goetz A., Jain A., Calvert V.S., Vishwakarma V., Wang D., Preet R., Cassel J., Summer R., Shaghaghi H., Pommier Y., Baechler S.A., Pishvaian M.J., Golan T., Yeo C.J., Petricoin E.F., Prendergast G.C., Salvino J., Singh P.K., Dixon D.A, & Brody J.R. (2021). The FDA approved anthelmintic Pyrvinium Pamoate inhibits pancreatic cancer cells in nutrient depleted conditions by targeting the mitochondria. Molecular cancer therapeutics, 20(11), 2166-2176.
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3

Quantitative Analysis of PD-L1 Secretion

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Cells were plated at 2×106 per P10 plate. Fresh media (6 ml) was added to the cells and collected after 24 hours. Collected media was spun at 4°C, 5 min, 1200 rpm to remove any cells and cell debris. After debris removal, the protein content in the media was concentrated 6 to 8-fold by using the 10K protein concentrator PES (Thermo Scientific) at 4°C. The protein concentrate was reconstituted in Cell Lysis Buffer (Cell Signaling Technology) and analyzed by Western blot.
Antibodies, Reagents, and Drugs. HIF1A, Lamin A/C, hPDL1, anti-biotin HRP-linked, and anti-rabbit IgG1 HRP-linked antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA); Actin, HuR 3A2, PDL1, donkey anti-goat IgG-HRP, goat anti-mouse IgG1-HRP antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA); the mPDL1/B7-H1 antibody was from B&D systems (Minneapolis, MN, USA); the alpha Tubulin antibody was from Sigma (Sigma-Aldrich). Mouse PDL1 ELISA Kit was purchased from Boster (Pleasanton, CA, USA). MLN4924 was purchased from Active Biochem (Kowloon Bay, Hong Kong); the PD1/PDL1-inhibitor-1 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).
Filippova N., Yang X., An Z., Nabors L.B, & Pereboeva L. (2018). Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment. Journal of cancer science & therapy, 10(8), 190-197.
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4

Profiling Protein Expression and Modifications

Cited 1 time
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Cytoplasmic and nuclear extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo-Scientific) as per manufacturer’s instructions. Total protein extracts were isolated and immunoblotting was performed as previously described (18 (link)). Primary antibodies used are HuR (3A2, 1:10,000; Santa Cruz Biotechnology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:10,000; Cell Signaling Technology), poly ADP-ribose polymerase (PARP-1; 1:1000; Santa Cruz Biotechnology), PAR (1:1000; Trevigen), PARG (1:1000; Millipore, Abcam), Caspase-3 (1:1000; Cell Signaling Technologies), γH2AX (1:1,000; Millipore), Lamin A/C (1:1,000; Cell Signaling Technology). The membranes were scanned and quantified using Odyssey Infrared Imaging System (LI-COR Biosciences).
Chand S.N., Zarei M., Schiewer M.J., Kamath A.R., Romeo C., Lal S., Cozzitorto J.A., Nevler A., Scolaro L., Londin E., Jiang W., Meisner-Kober N., Pishvaian M.J., Knudsen K.E., Yeo C.J., Pascal J.M., Winter J.M, & Brody J.R. (2017). Post-transcriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors. Cancer research, 77(18), 5011-5025.
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5

Immunohistochemical Profiling of Mouse Brain

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Upon complete anesthesia by isoflurane, mice were transcardially perfused with 15 mL ice-cold PBS at ~ 7 mL/min, followed by 20 mL ice-cold 4% paraformaldehyde (PFA) at ~ 4 mL/min. Brains were extracted and post-fixed in 4% PFA overnight at 4 °C. Fixed brains were coronally sliced by 2 mm on a Precisian Brain Slicer (Braintree Scientific), followed by dehydration in 70% EtOH for 24 h. Specimens were processed on a Leica Tissue Processor before embedding in paraffin blocks. For immunohistochemistry, 8 μm sections were stained with the following antibodies: HuR (3A2, Santa Cruz Biotechnology) at 1:200, Iba-1 (Wako) at 1:50, ki67 at 1:1000, MPO (Abcam) at 1:50 and CD4 (Abcam, 1:1,000).
Wang J., Leavenworth J., Hjelmeland A.B., Smith R., Patel N., Borg B., Si Y, & King P.H. (2019). Deletion of the RNA regulator HuR in tumor associated microglia and macrophages stimulates antitumor immunity and attenuates glioma growth. Glia, 67(12), 2424-2439.
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6

Quantitative Analysis of PD-L1 Secretion

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Cells were plated at 2×106 per P10 plate. Fresh media (6 ml) was added to the cells and collected after 24 hours. Collected media was spun at 4°C, 5 min, 1200 rpm to remove any cells and cell debris. After debris removal, the protein content in the media was concentrated 6 to 8-fold by using the 10K protein concentrator PES (Thermo Scientific) at 4°C. The protein concentrate was reconstituted in Cell Lysis Buffer (Cell Signaling Technology) and analyzed by Western blot.
Antibodies, Reagents, and Drugs. HIF1A, Lamin A/C, hPDL1, anti-biotin HRP-linked, and anti-rabbit IgG1 HRP-linked antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA); Actin, HuR 3A2, PDL1, donkey anti-goat IgG-HRP, goat anti-mouse IgG1-HRP antibodies were from Santa Cruz Biotechnology (Dallas, Texas, USA); the mPDL1/B7-H1 antibody was from B&D systems (Minneapolis, MN, USA); the alpha Tubulin antibody was from Sigma (Sigma-Aldrich). Mouse PDL1 ELISA Kit was purchased from Boster (Pleasanton, CA, USA). MLN4924 was purchased from Active Biochem (Kowloon Bay, Hong Kong); the PD1/PDL1-inhibitor-1 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA).
Filippova N., Yang X., An Z., Nabors L.B, & Pereboeva L. (2018). Blocking PD1/PDL1 Interactions Together with MLN4924 Therapy is a Potential Strategy for Glioma Treatment. Journal of cancer science & therapy, 10(8), 190-197.
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