Fig. 6.
Inhibition of RIDD by 4μ8C. (A) Autoradiograph of in vitro transcribed 32P-labeled mouse Xbp1 or Insulin2 (Ins2) mRNA separated by electrophoresis after incubation with the indicated concentration of IRE1 in the absence or presence of 4μ8C (10 μM). (B) RNase L activity measured with a FRET assay in the presence of 2–5A cofactor and in the presence or absence of the indicated concentrations of 4μ8C or Sunitinib (45). (C) Expression levels determined by qPCR of known IRE1 RIDD target genes following 6 h of treatment with tunicamycin (2.5 μg/mL) in the presence or absence of coapplied 32 μM 4μ8C (mean ± SEM, n = 2). (D) Plot of mRNA level of the RIDD target gene Scara3 in tunicamycin-treated cells exposed simultaneously to the indicated concentrations of 4μ8C (mean ± SEM, n = 2) for 6 h. (E) Immunoblot of puromycinylated proteins recovered from the membrane fraction of semipermeabilized wild-type and Perk-/- mutant MEFs that had been exposed to thapsigargin (500 nM) or tunicamycin (2.5 μg/mL) in the absence and presence of 32 μM 4μ8C for the indicated times. The intensity of the signal, integrated over the surface of each lane, is plotted under the image of the immunoblot.