ISG15/UCRP Antibody (539442) [Unconjugated]

Western blot shows lysates of MCF-7 human breast cancer cell line and HT-29 human colon adenocarcinoma cell line. PVDF membrane was probed with 0.5 µg/mL of Mouse Anti-Human ISG15/UCRP Monoclonal Antibody (Catalog # MAB4845) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for ISG15/UCRP at approximately 15 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

Simple Western lane view shows lysates of MCF‑7 human breast cancer cell line and HT‑29 human colon adenocarcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for ISG15/UCRP at approximately 20 kDa (as indicated) using 5 µg/mL of Mouse Anti-Human ISG15/UCRP Monoclonal Antibody (Catalog # MAB4845) . This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

ISG15 is necessary for autocrine IFNAR1-mediated control of DV replication. (C, D) A549 infected with 20 DV PFU. At 36 hpi, harvested, cell lysates analyzed by immunoblotting (C). Error bars represent mean ± SD. Results are representative of two independent experiments. Data was analyzed by unpaired t test. Image collected & cropped by CiteAb from the following open publication (//www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

ISG15 is necessary for autocrine IFNAR1-mediated control of DV replication. (A) A549 primed with IFN alpha 2b (100 IU/ml) for 12 h, washed three times with DPBS & allowed to rest. harvested at the indicated time point & cell lysates analyzed by WB with the corresponding antibodies. Results are representative of two independent experiments. Image collected & cropped by CiteAb from the following open publication (//www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

ISG15/UCRP was detected in immersion fixed paraffin-embedded sections of Human Endometrial Cancer using Mouse Anti-Human ISG15/UCRP Monoclonal Antibody (Catalog # MAB4845) at 0.5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using VisUCyte Antigen Retrieval Reagent-Basic (Catalog # VCTS021). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in cancer cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

ISG15 counteracts DV IFN-I evasion. (A) A549 WT and ISG15 KO were infected with 20 DV PFU. Cells were harvested at the indicated time points after infection (hpi) and cell lysates were analyzed by Western blot using STAT2 and Actin antibodies. (B–D) A549 WT and ISG15 KO immunofluorescence assay (IFA) 36 hpi for cellular IFIT3 and flavivirus E protein expression (D). Percentage of IFIT3 (B) and DV (C) positive cells per foci were quantified by ImageJ software and analyzed using unpaired t test with Welch’s correction when appropriate. Displayed images were acquired with a Leica DMI6000 B microscope. (E) A549 cells were infected with DV at MOI 0.01. At 36 hpi, cells were fixed, permeabilized and stained for flavivirus E protein. Cell lysates were analyzed by Western blot with the indicated antibodies before (upper panel) and after (bottom panel) cells were sorted by fluorescence-activated cell sorting (FACS) based on E protein expression. U, uninfected; B, bystander; I, infected. Error bars represent mean ± SD. Results are representative of two independent experiments. Statistical analyses were performed using Prism 8 (GraphPad Software). p values ***<0.001. Image collected and cropped by CiteAb from the following open publication (//www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

PKR deletion does not affect the innate immune response or cell viability. A549 WT or PKR−/− cells infected with DENV4 (MOI 2) or ZIKV (MOI 3) and harvested at 24 h.p.i. for analysis. (A) Quantification by RT-qPCR of IFN beta , IFN lambda , ISG15, and TNF-alpha gene expression. Relative expression calculated by 2− delta delta Ct methods using mock cells as a reference. (B) Immunoblot analysis of cell extracts resolved in denaturing SDS-PAGE. Representative image of two independent experiments. (C) Flow cytometry analysis for quantification of living cells by staining with Zombie NIR viability dye. Mock WT cells set as 100% reference. In the column charts, bars represent the means ± standard error of the mean from three independent experiments. Statistical analysis was performed by paired t test comparing the two cell lineages under the same conditions. *P ≤ 0.05. ns/no markup, no statistical difference. Image collected and cropped by CiteAb from the following open publication (//pubmed.ncbi.nlm.nih.gov/37732809), licensed under a CC-BY license. Not internally tested by R&D Systems.

ISGylation is not sufficient to restrict DV spread. (A) ISGylation profile of A549 WT and HERC5 KO cells by Western blot. Cells were primed with IFN alpha 2b (100 IU/ml) for 24 h and cell lysates were analyzed with an ISG15 antibody. (*) indicates antibody unspecific band. (B, C) A549 cells were infected with 20 DV PFUs. At 36 hpi cells were fixed, permeabilized and stained for the flavivirus E protein. DV relative foci area (B) and the number of infected cells per foci (C) quantified by ImageJ software and analyzed by one-way ANOVA. Images were acquired with an Olympus IX83 inverted microscope. Error bars represent mean ± SD. Results are representative of three or more independent experiments. Statistical analyses were performed using Prism 8 (GraphPad Software). p values ****<0.0001. Image collected and cropped by CiteAb from the following open publication (//www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

• Reactivity: Hu
• Applications: WB, Simple Western, IHC
• Clone: 539442
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: LYOPH

ISG15/UCRP Antibody (539442) [Unconjugated] Summary

• Immunogen: E. coli-derived recombinant human ISG15/UCRP
  Gly2-Gly157
  Accession # ABW03539
• Specificity: Detects human ISG15/UCRP in direct ELISAs and Western blots.
• Source: N/A
• Isotype: IgG2b
• Clonality: Monoclonal
• Host: Mouse
• Gene: ISG15
• Purity Statement: Protein A or G purified from hybridoma culture supernatant

Applications/Dilutions

• Dilutions:
  • Immunohistochemistry 8-25 ug/mL
  • Simple Western 5 ug/mL
  • Western Blot 0.5 ug/mL

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.
• Preservative: No Preservative
• Concentration: LYOPH
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in sterile PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for ISG15/UCRP Antibody (539442) [Unconjugated]

• G1P2
• G1P2interferon-induced 17-kDa/15-kDa protein
• IFI15
• IFI15hUCRP
• interferon, alpha-inducible protein (clone IFI-15K)
• Interferon-induced 15 kDa protein
• Interferon-induced 17 kDa protein
• interferon-stimulated protein, 15 kDa
• IP17
• ISG15 ubiquitin-like modifier
• ISG15
• Ubiquitin cross-reactive protein
• ubiquitin-like protein ISG15
• UCRP
• UCRPIP17

Background

ISG15 (interferon-induced 15 kDa protein; also known as IP17) is a 15 kDa ubiquitin family protein that is expressed by a wide variety of cell types. ISG15 is induced by type I IFN, and like ubiquitin, becomes conjugated to intracellular proteins. Unlike ubiquitin, it does not target proteins for degradation. Instead, it is part of the antivirus response. Human proISG15 is 165 amino acids (aa) in length. There are two ubiquitin-like domains (aa 2‑78 and 79‑157) and a propeptide (aa 158‑165) that is cleaved intracellularly. Select blood mononuclear cells secrete mature ISG15, which is chemotactic for neutrophils. Mature human ISG15 shares 66% aa sequence identity with mouse ISG15.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for ISG15/UCRP Antibody (MAB4845)(1)

Publication using MAB4845

• Lange C, Gouttenoire J, Duong F, Morikawa K, Heim M, Moradpour D Vitamin D receptor and Jak-STAT signaling crosstalk results in calcitriol-mediated increase of hepatocellular response to IFN-alpha. J Immunol, 2014-05-12;192(12):6037-44. 2014-05-12 [PMID: 24821973] (Western Blot, Human)

Bioinformatics

• Gene Symbol: ISG15
• Uniprot:
  • ABW03539()