In vitro IRE1α kinase activity assay

The kinase activity of IRE1α in the presence of fortilin was assayed in vitro as previously described⁴¹ using (a) [γ-³³P]ATP or (b) anti-phosphoserine antibody as follows. First recombinant human IRE1α (15 nM, SignalChem), MBP (20 µM, SignalChem), and various concentrations of fortilin were prepared in kinase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij35, 0.02 mg mL⁻¹ BSA, 0.1 mM Na₃VO₄, 2 mM DTT, and 1% DMSO) and incubated for 20 min at room temperature. The reaction was initiated by addition of [γ-³³P]ATP (10 µM), carried out for 2 h at room temperature, and terminated by spotting the reaction mixture onto phospho-cellulose P81 ion exchange filter paper (GE Whatman, Pittsburgh, PA, USA). The filter paper was washed three times with 1% phosphoric acid to remove unbound phosphate, placed in a scintillation tube containing scintillation cocktail, and subjected to scintillation counting. After subtracting background derived from control reactions lacking the kinase, kinase activity data were expressed as the percent kinase activity in test samples compared to reaction mixture with no fortilin. The data from two independent experiments were interpolated to a sigmoidal four parameter logistic curve, from which the half maximal inhibitory concentration (IC₅₀) of fortilin was derived (GraphPad Prism, La Jolla, CA, USA) (Fig. 3l). For the further assessment of in vitro IRE1α kinase activity, recombinant human IRE1α (0.5 µg, SignalChem) was pre-incubated with various concentrations of recombinant human fortilin (0–4 µg) in Kinase Assay Buffer (25 mM MOPS pH 7.2, 12.5 mM β-glycerol-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA, 0.25 mM DTT, 20 µM ATP) for 15 min at room temperature, followed by the addition of 2 µg of MBP (SignalChem) as a universal kinase substrate⁷⁶ to the reaction mixture. The reaction mixture was incubated at 30 °C for 45 min and subjected to SDS-PAGE and immunoblot analysis using anti-phosphoserine (EMD Millipore), anti-MBP (Santa Cruz), anti-fortilin (Abnova), and anti-phospho-IRE1α (Abcam) antibodies. The phosphorylation index was calculated by dividing the signal intensity of phosphorylated MBP by that of total MBP; results are expressed as A.U. Two independent experiments were performed with consistent findings (Supplementary Fig. ³F).