Fig. 2.

Establishment of HiBiT knock-in cells to monitor intrinsic ATF4 protein expression in Neuro2a cells. A, B) The schematic structure of the donor gene coding the C-terminal region of ATF4 fused with the HiBiT epitope and the strategy for establishing the HiBiT knock-in cells. An arrowhead indicates the integrated site. Dashed arrows indicate the PCR primer pairs used for the amplification of gene sequences around the HiBiT-integrated mouse ATF4 gene in the knock-in cells. C) Nucleotide sequences corresponding to the C-terminal region of the mouse ATF4 gene (top, NM_009716.3) and those edited by the present CRISPR/Cas9 system. The underlined italic letters and the three bold letters indicate the target sequence of the gRNA and PAM site, respectively. The bold letters in the second and third nucleotide sequence (#1, #2 and #3) indicates substituted nucleotide, respectively. The underlined large letters indicate the nucleotide sequences of the HiBiT epitope. The sequences of #2 and #3 around the ATF4 C-terminal region were identical. D) Wild-type (wt) Neuro2a and 1 and 2 (#1 and #2) clone cells in 12-well plates were treated with MG132 (MG, 10 μM) or vehicle for 12 h. After determination of the protein concentration of each lysate, equal amounts of cell lysate were separated with SDS-PAGE and transferred onto a PVDF membrane. Expression levels of HiBiT-derived signals (left), ATF4 and G3PDH (right) were detected as described in the Materials and methods section.