Fig 1: PKA inhibitor abolished phosphorylation of PKA downstream signaling induced by Y1 receptor deficiency. BMSCs were incubated in osteogenic media for 5 days. (A) Expression levels of PKA, CREB, and RUNX2 were quantified by Western blot analysis. (B) Cell lysates were collected and fractionated into nuclear and cytosolic extracts. Expression levels of p-CREB and CREB were analyzed by Western blot analysis. LDH was used as a control for cytosol protein samples, and Lamin B was used as a control for nuclear protein samples. (C) CREB transcriptional activity was assessed by luciferase reporter assay in BMSCs treated with Y1 shRNA or H-89 for 5 days during osteoblast differentiation. The luciferase activity of the cell lysates was measured using Dual-luciferase reporter assay system with normalization to Renilla luminescence (N = 5). Data are presented as mean ± SD; *p < 0.05 compared to control. GAPDH: glyceraldehyde 3-phosphate dehydrogenase. con: cells cultured in osteogenic media. CREB-luc: cAMP-response element binding protein (CREB) luciferase reporter plasmid (data presented in A, B are representative; data presented in C are pooled).