Anti phospho creb antibody

The dissection and homogenization of the TG and Western blot analysis were performed as described in detail previously [32 (link)]. The primary antibodies were as follows: anti-Nav1.7 antibody (1:1000, 20257-1-AP, Proteintech, USA), anti-COX-2 antibody (1:1000, 12282s, Cell Signaling Technology, USA), anti-phospho-CREB antibody (1:1000, 9198s, Cell Signaling Technology, USA), anti-CREB antibody (1:1000, 9191s, Cell Signaling Technology, USA), anti-IL-1β antibody (1:500, YR0913021, R&D systems, USA), anti-glial fibrillary acidic protein (GFAP; a marker of glial activation) antibody (1:1000, P14136, Cell Signaling Technology, USA), and anti-β-actin antibody (1:10000, TA-09, ZSGB-BIO, China). The densities of the bands were quantified using the NIH ImageJ 1.38 software (NIH, Bethesda, MD, USA) and expressed as fold change of the control group after normalization to β-actin or CREB.

Protein extraction from cultured cells or tissues was carried out using RIPA lysis buffer (Beyotime) containing 1 mM PMSF and a protease and phosphatase inhibitor cocktail (Beyotime). Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime) in accordance with the manufacturer’s instructions. Equivalent amounts of proteins (40 μg) from each sample were separated via 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a Poly vinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was then immersed within a 5% nonfat-dried milk and Tris-Buffered Saline Tween-20 (TBST) solution for 2 h at room temperature. Afterward, the membrane was incubated with primary anti-CD38 antibodies (1:1000; Abcam), anti-PTEN antibodies (1:1000; Abcam), anti-CREB antibodies (1:500; Proteintech group, Wuhan, China), anti-phospho-CREB antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) or anti-β-actin antibodies (1:500; BOSTER Biological Technology). After an overnight incubation at 4°C, the membrane was washed with TBST and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:50000; BOSTER Biological Technology) for 2 h at 37°C. To develop protein bands, the membrane was incubated with enhanced chemiluminescence substrate (Applygen Technologies, Beijing, China) and exposed to x-ray film.