Pcreb

CH-I, No. 0190501–1, Zhitong Biopharma Co. Ltd., China; CBL, No. 1–21380, EVER Neuro Pharma GmbH, Austria;2,3,5-Triphenyltetrazolium chloride (TTC), No. T8877, SIGMA, USA; Hematoxylin-Eosin (HE) staining kit, No. G1120, Beijing Solarbio Science & Technology Co. Ltd., China; Colorimetric TUNEL apoptosis assay kit (C1091), Beyotime Biotechnology Co. Ltd., China; Rabbit anti-IgG MEK1/2 (#380797), pMEK1/2 (Ser217/221, #310050), ERK1/2 (#343830), pERK1/2 (Thr202/Tyr204, #301245), CREB (#383983), pCREB (Ser133, #380697), ZEN-BIOSCIENCE, China; Rabbit anti-mouse polyclonal antibody BDNF (Ab-DF6387), Affinity Co. Ltd., USA.

Cells were lysed using radioimmunoprecipitation assay (Solarbio) containing protease phosphatase inhibitor cocktail (Solarbio) for protein extraction. Protein lysates (20 μg) were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for antibody incubation. After blocked with Tris-buffered saline, 0.1% Tween 20 (TBST) containing 5% skim milk powder, the membrane was probed overnight with primary antibodies at 4°C and washed with TBST. After that, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Afterward, the protein bands were visualized using the ECL kit (Monad) and recorded with the Alpha-FluorChemQ imaging system.
The following primary antibodies were used: RUNX2 (sc-390351, Santa Cruz Biotechnology), OSX (ab209484, Abcam), CREB (381013, ZenBio), p-CREB (380697, ZenBio), JUNB (sc-8051, Santa Cruz Biotechnology), PKA (251816, ZenBio), CALCRL (bs-1860R, Bioss), RAMP1 (R25544, ZenBio), SHH (BF0146, Affinity), and glyceraldehyde-3-phosphate dehydrogenase (BS65483M, BIOGOT; 380626, ZenBio). The following secondary antibodies were used: goat anti-mouse immunoglobulin G (IgG) H&L [horseradish peroxidase (HRP)] (511103, ZenBio) and goat anti-rabbit IgG H&L (HRP) (511203, ZenBio).