Figure 6: Effects of kinase domain active site binders on murine IRE1α function.

(a) Analysis of oligomerization status. Velocity analytical ultracentrifugation analyses were performed on murine IRE1α preparations (labelled on left) at 18 μM protein concentration. See Supplementary Fig. 6 for full protein concentration series. ADP and JAK inhibitor I concentrations were 500 and 30 μM, respectively. SDS–PAGE analysis of murine IREα pre- and post-treatment with lambda phosphatase is shown in the right panel. (b) Chemical structures of ADP and JAK inhibitor I. (c) Effect of ADP on the RNase activity of murine IRE1α using a model single-hairpin substrate labelled with fluorescein. Reaction products were separated via PAGE, detected using a GE Healthcare Typhoon Variable Mode Imager (top panel) and quantified using ImageQuant 5 software (bottom panel). Shown is a representative profile. IC₅₀±s.d. was calculated for n=3. (d) Dose-response analysis of JAK inhibitor I on the RNase activity of murine IRE1α assayed as in c. Shown is a representative profile. EC₅₀±s.d. was calculated for n=3. (e) Dose-response analysis of JAK inhibitor I on the auto-kinase activity of murine IRE1α as assessed using P³²-γ-ATP. Quantification plot presented in lower panel represents the 10 min time point from auto-radiographs in the top panel. Shown is a representative profile. IC₅₀ was calculated for n=2 (two experiments performed in singlicate). (f) Effect of the indicated back-to-back dimer interface mutations on murine IRE1α RNase function, as assayed in Fig. 1b. (g) Effect of the indicated back-to-back dimer interface mutations on murine IRE1α auto-kinase function (phosphor image, top; quantification, bottom), as assayed in Fig. 1c.