Fig. 1.

Transient overexpression of HiBiT-tagged ATF4 in Neuro2a cells. A) Schematic structure of a HiBiT-tagged ATF4 construct. B) A mechanism of HiBiT-derived luciferase activity. C) Twenty-four hours after transfection with HiBiT-tagged ATF4 or pcDNA3.1 empty vector, cells were treated with MG132 (MG, 10 μM) or vehicle for an additional 12 h. After the cells were harvested and lysed with homogenization buffer, each lysate containing 1 μg protein was mixed with the same amount of reaction mixture containing recombinant LgBiT (rLgBiT) and furimazine in diluted HiBiT lytic buffer. After an incubation at 37 °C for 10 min, each luciferase activity in each sample was measured as described in the Materials and methods section. D) Equal amounts of cell lysate prepared in (C) were separated with SDS-PAGE and transferred onto PVDF membranes. Expression levels of HiBiT-derived signals, ATF4 and G3PDH were detected as described in the Materials and methods section.