Anti hur

Manufactured by Proteintech

Sourced in China

Anti‐HuR is a laboratory reagent used to detect and study the HuR (Human antigen R) protein, which is involved in the regulation of gene expression. The product is a highly specific antibody that can be used in various experimental techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and quantify the HuR protein in biological samples.

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Lab products found in correlation

Anti hsp90, by Proteintech (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Lsm 880, by Zeiss (2 mentions) Gel doc xr, by Bio-Rad (1 mentions) Anti vegf, by Proteintech (1 mentions) Anti β actin, by Beyotime (1 mentions) Anti α tubulin, by Beyotime (1 mentions) Anti cd81, by Proteintech (1 mentions) Ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions) Anti gapdh, by Beyotime (1 mentions) Anti cd63, by Abcam (1 mentions) Anti calnexin, by Abcam (1 mentions) Anti tsg101, by Abcam (1 mentions) Pannoramic scan, by 3DHISTECH (1 mentions) Protein a g magnetic beads, by Beyotime (1 mentions) Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions) Anti ubiquitin, by Proteintech (1 mentions) Anti usp5, by Proteintech (1 mentions) Ez magna rip kit, by Merck Group (1 mentions) Anti bcl 2, by Proteintech (1 mentions)

7 protocols using anti hur

Western Blot, Immunohistochemistry, and Immunoprecipitation Protocols

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Proteins in cells and tissues were extracted with RIPA lysis buffer (Thermo Fisher). Serum proteins were extracted with Serum Protein Extraction Kit (Qcheng Bio, China). Western blot assays were performed according to details previously reported [23 (link)]. the immuno-complexes were detected with ECL Western Blotting Substrate (Thermo Fisher), visualized with BIO-RAD (BIO-RAD Gel Doc XR+, USA). The following antibodies were used (11000): anti-β-actin (Beyotime, AF0003); anti-α-tubulin (Beyotime, AF0001); anti-GAPDH (Beyotime, AF0006); anti-HSP90 (Proteintech, 60,318–1-Ig); anti-HUR (Proteintech, 11,910–1-AP); anti-EIF2S1 (Proteintech, 11,170–1-AP); anti-VEGF (Proteintech, 19,003–1-AP); anti-Calnexin (Abcam, ab92573); anti-CD63 (Abcam, ab134045); anti-TSG101 (Abcam, ab125011); anti-CD81 (Proteintech, 66,866–1-Ig); anti-STUB1 (Proteintech, 55,430–1-AP).
IHC, IF and IP was performed as previously described [24 (link)]. IHC was performed with antibodies against HUR and VEGF (Proteintech, 1:200). IF was performed with antibody against CD31 (Proteintech, 11,265–1-AP, 1:200). The images were scanned by Pannoramic SCAN (3DHistech, Hungary). IP was performed with anti-HSP90 antibody (Proteintech, 1:200) and appropriate control IgG (Merck Millipore), and the immunoprecipitate was then collected by centrifugation and analysed by SDS-PAGE.

Xie M., Yu T., Jing X., Ma L., Fan Y., Yang F., Ma P., Jiang H., Wu X., Shu Y, & Xu T. (2020). Exosomal circSHKBP1 promotes gastric cancer progression via regulating the miR-582-3p/HUR/VEGF axis and suppressing HSP90 degradation. Molecular Cancer, 19, 112.

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Validating HuR-Mediated Protein Interactions

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A co‐immunoprecipitation kit with Protein A + G Magnetic Beads (Beyotime, CN) was used to validate the interaction between HuR protein and β‐catenin as well as G3BP1. In brief, magnetic beads were coated with 5 μg of antibodies, including anti‐HuR (Proteintech, CN) and anti‐immunoglobulin G (IgG) (Beyotime, CN), for 30 min at room temperature. The cell lysate from 2 × 10⁷ cells was incubated with antibody‐coated magnetic beads overnight at 4°C to form beads antigen–antibody complexes. After washing away nonspecific proteins, the immunoprecipitated complexes are eluted and subjected to western blotting analysis to study protein–protein interactions.

Huai Y., Wang X., Mao W., Wang X., Zhao Y., Chu X., Huang Q., Ru K., Zhang L., Li Y., Chen Z, & Qian A. (2024). HuR‐positive stress granules: Potential targets for age‐related osteoporosis. Aging Cell, 23(3), e14053.

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Immunoprecipitation of RNA-Binding Proteins

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The MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore) was used according to the manufacturer’s instructions. The cell lysates were incubated with beads coated with 5 μg of antibody against Argonaute-2 (AGO2) (Abcam, MA, USA), anti-HSP90 (Proteintech) or anti-HUR (Proteintech) and control IgG with rotation at 4 °C overnight. Next, total RNA was retrieved for the detection of circRNAs and miRNA expression by qRT-PCR.

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Immunoprecipitation and Western Blot Analysis

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The growth medium of adherent cells in a 10 cm cell culture dish was removed by aspiration and washed once with PBS. The cells were then lysed with 1 mL IP buffer, mixed with 2 μg anti‐HuR (Proteintech, 11910‐1‐AP) and incubated with rotation overnight at 4°C. Protein A/G Agarose was fully resuspended in 20 μL buffer A and shaken slowly. IP complex was washed in IP buffer five times and 20 μL of 1 × SDS‐PAGE loading buffer was added and mixed. The pellet was vortexed and then centrifuged to concentrate the sample at the bottom of the tube. Western blot analysis was performed using antiubiquitin (Proteintech, 10201‐2‐AP) and anti‐USP5 (Proteintech, 10473‐1‐AP).

Xiao M., Liu J., Xiang L., Zhao K., He D., Zeng Q., Zhang Q., Xie D., Deng M., Zhu Y., Zhang Y., Liu Y., Bo H., Liu X., Chen X., Gong L., Bao Y., Hu Y., Cheng Y., Deng L., Zhu R., Xing X., Zhou M., Xiong W., Zhou Y., Zhou J., Li X, & Cao K. (2020). MAFG‐AS1 promotes tumor progression via regulation of the HuR/PTBP1 axis in bladder urothelial carcinoma. Clinical and Translational Medicine, 10(8), e241.

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Immunofluorescence Imaging of HuR Protein

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MRC-5 cells were seeded on the cell slides in a 24-well plate, fixed with 4% tissue cell fixative for 30 min, and punched with 0.3% TritonX-100. The slides were washed with 1 × PBS, blocked with goat serum for 1 h, and incubated with anti-HuR (1:1,000, Proteintech, China) at 4°C overnight. Fluorescently labeled secondary antibody was added, incubated at room temperature for 60 min in the dark, and then discarded. 200 μl DAPI was added to each well to stain cell nuclei for 6 min and washed with 1 × PBS. Finally, the anti-fluorescence quencher was dropped on the cell slides. All images were collected under a laser scanning confocal microscope (Zeiss LSM880, Germany).

Zhu Q., Wang J., Ji Y., Luan J., Yue D., Liu W., Li H., Zhang J., Qu G., Lv C, & Song X. (2022). Danshensu methyl ester enhances autophagy to attenuate pulmonary fibrosis by targeting lncIAPF–HuR complex. Frontiers in Pharmacology, 13, 1013098.

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RIP Assay for HuR Binding

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The RIP assay was performed using the EZ‐Magna RIP kit (Millipore, MA). Briefly, collected and incubated BUC cells (1 × 10⁷) with RIP lysis buffer. The precleared lysates were used for RIP with anti‐HuR (Proteintech, 11910‐1‐AP) and rabbit isotype control IgG antibodies. RNA was isolated and purified using an acid phenol/chloroform method.

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Protein Extraction and Western Blot Analysis

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Proteins were extracted from cells using RIPA buffer (Beyotime, Shanghai, China), and the protein concentration of samples was quantified using a BCA assay kit (Beyotime). Equal amounts of proteins were separated by SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (PVDF; Solarbio). The membranes were blocked with 5% skim milk powder and incubated with specific primary antibodies against the following proteins: anti-β-actin (1:1000; ZSGB-BIO, Beijing, China), anti-Bcl-2 (1:2000; Proteintech Group, Wuhan, China), anti-Bax (1:2000; Proteintech Group), anti-HuR (1:1000; Proteintech Group) and anti-IGF2BP1 (1:1000; Proteintech Group) at 4°C overnight. The membranes were then incubated with the appropriate secondary antibodies (ZSGB-BIO) and visualized with enhanced chemiluminescence reagents (ECL; Beyotime). Band intensities were quantified using Image-Pro Plus 5.02 software (Media Cybernetics, Bethesda, USA). β-Actin was used as the loading control.

Li Y., Miao H., Wei W., Tian J, & Chen J. (2022). Inhibitory effect of calycosin on breast cancer cell progression through downregulating lncRNA HOTAIR and downstream targets: HuR and IGF2BP1: Calycosin inhibits breast cancer growth by regulating HOTAIR. Acta Biochimica et Biophysica Sinica, 55(2), 225-236.

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