Ab220807

The L4-L6 dorsal root ganglions (DRGs) of rats from different groups were sampled at specific timepoints, 0, 3, 7, and 14 d following CCI surgery. After 3 washes with icy PBS, the dissected tissues were rapidly transferred into the RIPA lysis buffer (#20-188, 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, and 10 mM EDTA) containing protease inhibitor (#I3786), all from [17]. Total protein was determined by a BCA kit (#P0012, Beyotime, Shanghai, China). For each sample 30 μg protein was loaded, then separated on 10% SDS-PAGE gel, and transferred to a PVDF membrane (#IPVH00010, Merk Millipore, Billerica, MA). Then the membrane was blocked with 5% bovine serum albumin for 2 h at room temperature and incubated with primary antibodies, rabbit anti-Sirt1 (#ab220807, Abcam, Cambridge, UK), mouse anti-Nav1.7 (#ab85015, 1:500, Abcam), and rabbit anti-GAPDH (#ab9485, 1:2000, Abcam, Cambridge, MA, USA), at 4°C overnights. After 2-h incubation at room temperature with corresponding horseradish peroxidase-conjugated secondary antibodies (#AB6721 and #ab6728, 1:3000, Abcam), the targeted proteins were detected using Pierce ECL Western Blotting reagents (#32,106, ThermoFisher, Rockford, IL, USA) and imaged using FluorChem E (Alphalmager Proteinsimple, San Jose, California). GAPDH served as the inner control.

Cell lysates were subjected to Western blotting analysis according to standard protocols. After being blocked, the membranes were incubated overnight at 4°C with primary antibodies against SIRT1 (Abcam, ab220807), acetyl-P65 (Abcam) and IκBα (Cell Signaling Technology). GAPDH (Cell Signaling Technology) was used as a normalization control. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology). Blots were developed with enhanced chemiluminescence substrate (Millipore), and detection was performed using LAS-4000 (GE Healthcare).

Cultured cells were collected by trypsinization, and then lysed with enhanced radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor (Boster, Wuhan, China), and then the protein concentration was determined by bicinchoninic acid (BCA) protein quantitative kit (Boster). The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane and blocked with 5% bovine serum albumin at room temperature. Then diluted primary rabbit antibodies (SIRT1, ab220807; KLF4, ab106629; MMP2, ab97779; β-actin, ab8227; 1: 500; Abcam, Cambridge, MA, USA) were added into the membrane, followed by incubation at 4°C overnight. Next, horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (ab205719; 1: 2000; Abcam) was used to incubate the membrane for 1 hour at room temperature. The enhanced chemiluminescence solution (EMD Millipore, Bedford, MA, USA) was used to incubate the membrane at room temperature for 1 minute, after which it was then sealed with the plastic wrap, followed by X-ray film exposure for 5-10 minutes, development and fixation. Image J software was used to quantify the gray intensity of each band in Western blot analysis image, and β-actin was used as internal reference.