4μ8C (IRE1 Inhibitor III) is a highly selective and potent inhibitor of IRE1α ribonuclease (RNase) activity (IC₅₀ ≈ 6–10 μM). By forming a covalent bond with the RNase active site of IRE1α, 4μ8C specifically inhibits its ribonuclease activity, thereby blocking the splicing of XBP1 mRNA. 4μ8C can be used in research on endoplasmic reticulum stress, the unfolded protein response, immune regulation, and tumor and metabolic diseases.

IRE1 Rnase:76 nM

In Vitro IRE1 RNase and RIDD Assays: Analysis of radiolabeled Xbp1 substrate cleavage is performed as previously except that mammalian IRE1 reaction buffer is used. In vitro RIDD substrates are synthesized by in vitro transcription using the T7-MAXIscript Kit in the presence of 32P ATP or Cy5-UTP on templates isolated by RT-PCR from mouse Min6 cells (Ins2) or PCR from cloned XBP1 cDNA. The resulting products are gel purified to obtain full-length substrate. Reactions are then separated by 15% UREA-PAGE for analysis by phosphorimaging or by near-infrared imaging using the LI-COR Odyssey scanner.

Cells are seeded in phenol red-free cell culture medium in 96 or 24 well dishes at a density of 5 × 103 or 5 × 104 cells per well, respectively. Cultures are incubated for 16 h before treatment with 4μ8C for 24 h. Cultures are then analyzed by the addition of 200 μM WST1 and 10 μM phenazine metho-sulfate. After development of the reagent for 2 h at 37°C, the hydrolyzed dye is detected by absorbance at 450 nm, after subtracting background and absorbance at 595 nm. Alternatively, cell viability is determined by staining of the adherent culture with crystal violet. Quantitation of the dye uptake is analyzed by extensive washing of the stained cells with water and solublization of the crystal violet in methanol followed by absorbance measurements at 595 nm. (Only for Reference)

IRE1 Inhibitor III

DMSO: 33.33 mg/mL (163.24 mM), Sonication is recommended.