Anti p p65

The whole cells were washed and lysed in RIPA buffer (89900, Thermo Scientific) supplemented with PMSF (phenylmethylsulfonyl fluoride) and protease inhibitors. Protein concentrations were determined with a BCA assay kit (Pierce, Rockford, IL, USA). Then the lysates were mixed with loading buffer, analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocked with 5% skim milk in TBST at room temperature for 1 h, the membranes were incubated with different primary antibodies, including anti-DDX54 (1:1000, Proteintech, China), anti-P65, anti-p-P65, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, (1:1000, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (1:5000, Yeason, China) in 5% milk/TBST buffer at 4°C overnight, and then probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h. After washing three times with TBST, the membrane was developed with enhanced chemiluminescent plus substrate (Merck Millipore, Billerica, MA, USA), and the signal was recorded by Fluorchem E System (ProteinSimple, Santa Clara, CA, USA).

First, hBMMSCs were washed with cold PBS softly for three times. After that, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phosphatase inhibitor (Roche) and 2% protease inhibitor cocktail (Roche). After collecting and centrifugation of the cell lysate at 13362g at 4 °C for 30 min, supernatants were carefully transferred to new tubes, and the BCA protein assay kit was used to determine the protein concentrations. Thirty-five-microgram total protein of each sample was subjected to 10% SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) after electrophoresis. The membrane was then blocked with 5% nonfat milk and then incubated with anti-IκBα (Abcam, Cambridge, UK), anti-p-IKK, anti-p-IκBα, anti-p-P65, anti-P65, anti-RUNX2, or anti-GAPDH (Cell Signaling Technology, Beverly, MA, USA) in TBS at 4 °C overnight. After that, the membrane was washed with Tris-buffered saline-Tween 20 (TBST) buffer for 3 times and then incubated with goat anti-rabbit IgG (Abcam). After that, the membrane was washed with TBST buffer again. Lastly, an ECL Western blot kit (CWBIO) was used to visualize the bands.