Creb antibody

For ChIP assay, approximately 1 × 10⁷ cells were harvested in medium and fixed with 1% formaldehyde. Glycine solution was added at a final concentration of 0.125 M to quench unreacted formaldehyde. Fixed cells were collected by spinning at 1500 rpm for 5 min. ChIP assays were performed using a ChIP Assay Kit (Cell Signaling Technology, Inc, USA) according to the manufacturer's instructions. IgG antibody was from the ChIP Assay Kit. CREB antibody was obtained from (Cell Signaling Technology, Inc, USA). Quantification of immunoprecipitated DNA was performed using qRT-PCR with LightCycler 480 SYBR Green I Master (Roche, US). The primers shown in Table 3 were designed according to the promoter region of SKA2 gene. Values derived from three independent experiments were normalized by background signals (IgG) and presented as percentage of Input chromatin (% Input) [37 (link), 38 (link)].

For HPLC analysis, esculin (EC, purity 98%) and esculetin (ECT, purity 98%) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Fraxin (FR, purity 98.9%) and formic acid (analytical reagent grade) were purchased from Merck KGaA (Darmstadt, Germany). For animal experiments, reserpine (purity 98%), esculin (purity 98%), esculetin (purity 98%), and fluoxetine (FXT, purity 98% in thin layer chromatography) were supplied by Sigma-Aldrich. Fraxin (purity 98%) was supplied by InterPharm Corporation (Koyang-si, Gyeonggi-do, South Korea). For anesthesia, tiletamine/zolazepam was supplied by Virbac (Zoletil 50; Cedex, France). For western blot analysis, actin antibody was supplied by Sigma- Aldrich. BDNF antibody was supplied by Abcam plc. (Cambridge, United Kingdom). CREB antibody and phosphorylated CREB (pCREB) antibody were supplied by Cell Signaling Technology (Danvers, MA, United STates). For immunofluorescence analysis, BDNF antibody was supplied by Abcam plc, pCREB antibody by Cell Signaling Technology, and NeuN antibody by Merck KGaA.

Sinomenine (No. 2000058, purity 94.9%) was bought from Hunan Zhengqing Pharmaceutical Co., Ltd., China. Morphine (30 mg/mL) was obtained from the drug supply station of the People’s Liberation Army, China. Naloxone, NMDA, and PKH67 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospho-NMDAR1 (Ser897), NMDAR1, p-CAMKII (Ser286), CAMKII, and p-CREB (Ser133) antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA). CREB antibody and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Calcium fluorescent probe Fluo-8 AM, GFAP, TSG101, CD81, and CD63 antibodies were bought from Abcam (Cambridge, UK). Cyclic-AMP direct immunoassay kit (colorimetric) was obtained from BioVision (Milpitas, CA, USA). The chemiluminescence reaction kit (ECL) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).