4μ8C (IRE1 Inhibitor III)(IC50=76 nM) is an effective and specific IRE1 Rnase inhibitor.

IRE1 Rnase:76 nM

4μ8C通过阻断其底物(RIDD)进入IRE1的活性部位，从而有选择性地失活Xbp1的剪接和IRE1介导的mRNA降解，进而诱导ER应激，但未观察到可测量的急性毒性。[1] 作为IRE1抑制剂，4μ8C同时阻止了来自CD4+ T细胞的IL-4、IL-5和IL-13的产生。[2]

4μ8C可逆转ER应激依赖的多个已知RIDD靶标的丧失，其半最大有效浓度（EC50）约为4μM，与XBP1靶基因激活的抑制作用相近[1]。

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)