Nb100 56875

Total cell lysates were isolated using cell lysis buffer (RIPA buffer: Cell Signaling Technology #9806, Danvers, MA) containing protease inhibitor cocktail (Roche, Nutley, NJ). Protein concentrations were determined by BCA assay (Sigma-Aldrich, St. Louis, MO). Proteins were separated by SDS-PAGE and transferred from gels to 0.2 μm nitrocellulose membranes (Pall Corporation, Washington, NY). The nitrocellulose membrane was further incubated overnight at 4°C with rabbit anti-VGLL3 (1:1000, Novus Biologicals, NB100-56875, Centennial, USA) and rabbit anti-GAPDH (1:2000, Novus Biologicals, 4650S, Centennial, USA). After that membranes were incubated with HRP-conjugated anti-Rabbit IgG (1:1000, Cell Signaling Technology, 7074S, Danvers, MA) secondary antibody for 1 hour at RT, protein bands were visualised using western blotting luminol reagent (Santa Cruz Biotechology, Inc., Dallas, Texas).

Cells were collected in 1X PBS and spin down (250 g, 5 min) and pellet stored at -80 °C for further use. Whole cell lysates were prepared from cells by adding cell lysis RIPA buffer, complemented with phosphatase inhibitors (Roche) and kept 30 min on ice. For each sample, 30 μg of protein was resolved in a 4-15% gradient mini-protean precast polyacrylamide gels (BioRad) and transferred to nitrocellulose membranes (Whatman, GE Healthcare) for 1 h at 4 °C. After blocking for 1 h with 5% skim milk in PBST (0.1% Tween-20 in PBS), the membranes were incubated overnight at 4 °C with primary antibodies diluted in 5% milk. The following primary antibodies were used: TRF2, mouse monoclonal, (NB100-56506, 1:1000, Novus Biologicals); TRF2, rabbit monoclonal, (NB110-57130, 1:5000, Novus Biologicals); FOXO3a, rabbit monoclonal (clone 75D8, 1:1500, Cell Signaling Technology); GAPDH, rabbit polyclonal (1:2000, NB100-56875, Novus Biologicals). The membranes were then rinsed three times in PBST for 10 min and incubated 1 h at room temperature with appropriate secondary antibodies diluted (1:5000) in 5% PBST-milk (e.g. anti-mouse HRP IgG and anti-rabbit HRP IgG, Vector Labotratories). Membranes were developed using the Luminata Forte HRP substrate (Millipore) and exposed in the Fusion Solo apparatus (Vilbert Lourmat).

Chondrocytes were lysed with immunoprecipitation lysis buffer (Sigma‐Aldrich, MO) to extract total protein. BCA Protein Assay Kit (P0010, Beyotime, China) was used for the quantification of total protein. Protein extracts were subjected to 10% SDS‐PAGE, and transferred to polyvinylidene fluoride membranes. After blockade of 3% BSA, the membranes were incubated with primary antibodies FoxO3 (ab109629, Abcam, UK, 1:1000), COL2A1 (NB‐600‐844, Novus, MO, 1:1000), Sox9 (ab185966, Abcam, UK, 1:5000), ACAN (A11691, ABclonal, China, 1:500), ADAMTS5 (ab41037, Abcam, UK, 1:1000), Runx2 (ab236639, Abcam, UK, 1:1000), Bcl‐2 (ab32124, Abcam, UK, 1:1000), Bax (ab32503, Abcam, UK, 1:1000), PCNA (ab92552, Abcam, UK, 1:1000), and GAPDH (NB100‐56875, Novus, MO, 1:5000) at 4°C for 12 hours, and incubated with HRP goat anti‐rabbit IgG (AS014, ABclonal, China, 1:2000). The membrane was detected using BeyoECL Star (P0018AS, Beyotime, China). GAPDH was used as the internal reference.