Fig. 2: G6374 promotes a stable association between IRE1 and VHL. | Nature Communications

Fig. 2: G6374 promotes a stable association between IRE1 and VHL.

From: Chemically-induced degradation of the endoplasmic-reticulum stress sensor IRE1 by a VHL-recruiting chimera

Fig. 2

a co-IP of endogenous VHL with endogenous IRE1 from AMO1 cells upon addition of a titration of G6374 and competition with an IRE1 kinase inhibitor, G5758. Samples were analyzed by IB for IRE1, VHL, and GAPDH. b, c NanoBRET assay with over-expressed IRE1 and VHL in HEK293T cells with a titration of G6374 (n = 3 biological replicates, mean ± SEM). EC50 indicates the degrader concentration that achieves 50% maximal binding (as a readout of the NanoBRET ratio) (b). DC50 indicates the concentration at which 50% of the maximal achievable IRE1 depletion is observed; Dmax indicates the maximal percent depletion of IRE1 as compared to baseline (as a readout of the NanoLuc signal) (c). d Size-exclusion chromatography (SEC) of the IRE1:G6374:VCB complex or IRE1 alone. e Coomassie Blue staining of fractions from the peaks in (d) on SDS-PAGE gels. f Fluorescence polarization displacement assay performed with a titration of G6374 in the absence or presence of saturating IRE1 concentration (2.4 µM) to VCB bound with FAM-labeled HIF-1α peptide (n = 3 biological replicates, mean ± SEM).

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