Fig. 3.

Characterization of HiBiT-tagged ATF4 expression in Neuro2a cells. A) Wild-type Neuro2a cells in 12-well plates were treated with cycloheximide (CHX, 10 μg/ml), MG132 (MG, 10 μM) or vehicle for 6 h, and the expression levels of the indicated proteins were measured by immunoblot analysis as described in the Materials and methods section. B) After seeding #2 clone cells into a 96-well plate, the culture medium was replaced with OPTI-MEM, and the cells were treated with CHX (10 μg/ml), MG132 (10 μM) or vehicle for 6 h. After treatment, the cells were lysed with equal amounts of diluted HiBiT lytic buffer. Each lysate was mixed with equal amounts of reaction mixture containing rLgBiT and furimazine in diluted HiBiT lytic buffer and incubated at 37 °C. The HiBiT-derived luciferase activity of each sample was measured at the indicated time. The representative luciferase activities from control and MG132- or CHX-treated cells are indicated as circles, squares and triangles, respectively. C) #2 clone cells in a 96-well plate were treated with CHX (10 μg/ml), MG132 (10 μM) or vehicle for 1 or 6 h, and the cells were lysed as described above. After incubation of each lysate with reaction mixture at 37 °C for 10 min, luciferase activity was measured. Each value represents the mean ± SEM from 3 independent cultures. D, E) #2 clone cells in a 12-well plate were treated with tunicamycin (Tm, 1 μg/ml), MG132 (10 μM) or vehicle for 12 h. D) 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 rLgBiT and furimazine in diluted HiBiT lytic buffer. After the lysates were incubated at 37 °C for 10 min, luciferase activity in each lysate was measured. Each value represents the mean ± SEM from 6 independent cultures. E) Equal amounts of cell lysate prepared in (D) were separated with SDS-PAGE and transferred onto PVDF membranes. The expression levels of ATF4 and G3PDH were detected by immunoblot analysis. F) After seeding #2 clone cells into a 96-well plate, the culture medium was replaced with OPTI-MEM. The cells were treated with Tm (1 μg/ml, triangles) or vehicle (circles) for the indicated times, and they were lysed as described above. After incubation of each lysate with reaction mixture at 37 °C for 10 min, the luciferase activity of each lysate was measured. Each value represents the mean ± SEM from 3 independent cultures.