Cold cell lysis buffer

Cells were dissolved from triplicate samples (n = 3) in ice-cold cell lysis buffer (Beyotime) containing protease inhibitors; the protein concentration in cell extracts was quantified using a BCA protein assay kit (Beyotime). Equal amounts of protein from each extract were denatured and separated in a 10% polyacrylamide gel (Beyotime) and transferred by electrophoresis onto a nitrocellulose membrane (Thermo Fisher Scientific). The membrane was incubated with diluted primary antibodies against p16^INK4α, SIRT1, p38, p-p38, p21, p53, Erk1/2, p-Erk1/2, or α-tubulin at 4°C overnight, followed by the secondary antibody of horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit for 1 h at room temperature. SuperSignal West Pico Substrate and CL-XPosure Film (Thermo Fisher Scientific) were used for exposure. The intensity of the bands was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD).

Osteoclasts cultured on TCPS and ECM were collected and protein was extracted by ice-cold cell lysis buffer (Beyotime) containing protease inhibitors (Thermo Fisher Scientific). The protein concentration was measured using a BCA protein assay kit (Beyotime). Heat-denatured protein samples were separated by polyacrylamide gel electrophoresis (Beyotime) and then transferred onto nitrocellulose membranes (Thermo Fisher Scientific). After blocking with 5% non-fat dry milk for 1 h, the membranes were incubated with primary antibodies against IκB-α (phospho S32), IκB-α, p65, and GAPDH overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (H +L) and anti-rabbit IgG (H +L)-labeled secondary antibodies (Thermo Fisher Scientific). Specific immune complexes were visualized on a CL-XPosure Film (Thermo Fisher Scientific) using SuperSignal West Pico Substrate (Thermo Fisher Scientific) following the manufacturer’s instructions. The levels of each protein were compared after normalization against GAPDH as an internal control using the ImageJ software.