Human ISG15/UCRP Antibody

Detection of ISG15/UCRP in Human Endometrial Cancer.

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.

Detection of ISG15/UCRP by Western Blot

ISG15 is necessary for autocrine IFNAR1-mediated control of DV replication. (A) A549 primed with IFN alpha2b (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 (https://www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ISG15/UCRP by Western Blot

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 (https://www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ISG15/UCRP by Western Blot

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 deltaCt 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 (https://pubmed.ncbi.nlm.nih.gov/37732809), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ISG15/UCRP by Western Blot

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 alpha2b (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 (https://www.frontiersin.org/articles/10.3389/fimmu.2024.1331731/full), licensed under a CC-BY license. Not internally tested by R&D Systems.