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. 2021 Mar 3;4(2):780–789. doi: 10.1021/acsptsci.0c00218

Figure 9.

Figure 9

Homogenous high-throughput CETSA for IP6K1 inhibitors using HiBit-tagged IP6K1. (A) Linear relationship between luminescence signal detected in varous amount of cells expressing HiBit-tagged IP6K1. (B) Effect of heat treatment with or without 100 μM LI-2124 on the total luminescence signal detected in 12 500 cells. Cells were treated with or without LI-2124 followed by incubation at 37 or 51 °C. Total luminesence signals from HiBit-IP6K1 in the cells were detected by addition of lysis buffer containing detection reagents. (C) Effect of LI-2124 on HiBit-tagged IP6K1 in CETSA assay. HEK293T cells were transfected with plasmid expressing HiBit-IP6K1 (+) or empty vector (−). Cells were treated with LI-2124 for 30 min followed by incubation at 37 or 51 °C. Soluble fraction of the cell lysates were probed with anti-IP6K1 antibody by Western blot. The HiBit-tag in IP6K1 migrates almost identical to the endogenous IP6K1 in this SDS-PAGE. (D) Dose–response assay for IP6K1 inhibitors using HT-CETSA.