Figure 4.

Regulation of RAB27B by FOXO1 in BUMPT cells. BUMPT cells were transiently transfected with siRNA or plasmid. Cell lysates were collected 48 h later for immunoblot analysis. A: Predicted FOXO1 binding site in the mouse Rab27b gene promoter region. The 5' region of the mouse Rab27b gene was analyzed for FOXO1 binding sites using the JASPAR database (https://jaspar.genereg.net). The binding site with a JASPAR score >10 was listed. B: Conservation of the predicted FOXO1 binding site in mouse, rat, and human genomes. Bases in common among different species are colored red. C–E: Knockdown of FOXO1 decreased RAB27B expression. BUMPT cells were transiently transfected with Foxo1 siRNA or scrambled sequence siRNA to collect cell lysates for immunoblot analysis. C: Representative immunoblots of FOXO1 and RAB27B. Densitometric analyses of FOXO1 (D) and RAB27B (E). β-Actin was used as the loading control. After normalization with β-actin, the protein level in the scramble group was arbitrarily set as 1, and the protein level in the siFoxo1 group was compared with that 1 to calculate the fold change. *P < 0.05, ***P < 0.001 (n = 3). F–H: Nonphosphorylatable, constitutively active FOXO1 increased RAB27B expression. BUMPT cells were transiently transfected with empty vector or the plasmid of nonphosphorylatable, constitutively active Foxo1 to collect cell lysates for immunoblot analysis. F: Representative immunoblots of FOXO1 and RAB27B. Densitometric analyses of FOXO1 (G) and RAB27B (H). β-Actin was used as loading control. After normalization with β-actin, the protein level in the empty vector group was arbitrarily set as 1, and the protein level in the Foxo1 overexpression group was compared with that 1 to calculate the fold change. *P < 0.05, **P < 0.01 (n = 3).