Fig. 2

Role of PKA / CREB pathway in ethanol-induced NMDAR expression and intracellular calcium overload. a SK-N-MC cells were treated with EtOH (200 mM) for various time (0–6 h). Cat-PKA, p-CREB (Ser 113) and CREB were detected by western blot. β-Actin was used as a loading control. n = 3. b Cells were pretreated with 14–22 amide (1 μM) for 30 min before EtOH treatment. Cat-PKA, CREB and p-CREB (Ser 113) were analyzed by western blotting. β-Actin was used as a loading control. n = 4. c Cells were treated with EtOH (200 mM) for 12 h. mRNA expressions of GRIN1, GRIN2A, GRIN2B, and GRIN2D were analyzed by quantitative real time PCR. Data were normalized by the ACTB mRNA expression level. n = 4. d Cells were exposed to EtOH (200 mM) for 0–24 h. NR1 and NR2B were detected by western blot. n = 4. e Cells were transfected with CREB siRNA or NT siRNA for 24 h prior to ethanol exposure for 12 h. NR1 and NR2B expressions were measured by western blotting. n = 3. *p < 0.05 versus control with NT siRNA transfection, #p < 0.05 versus EtOH with NT siRNA transfection. f Cells were exposed to EtOH for 0–24 h and then loaded with Fluo 3-AM (2 μM) for 30 min. The amount of intracellular calcium was measured by using flow cytometry. n = 3. g Cells were pretreated with MK-801 (10 μM) for 30 min prior to EtOH treatment, subsequently loaded with Fluo 3-AM (2 μM) for 30 min. n = 4. h The population of MitoSOX™-positive cells was measured by flow cytometry. n = 3. i Apoptotic cells were measured by annexin V/PI analysis assay. All data are presented as a mean ± S.E.M. n = 3. All blot images are representative. *p < 0.05 versus control, ^#p < 0.05 versus EtOH