Sc 126

Cells were directly lysed in 2× SDS loading buffer and boiled for 15 min. Proteins were separated by SDS–PAGE, transferred to PVDF membrane, and probed with antibodies specific for, p-AKT (1:2000 dilution, s473, 4060, Cell Signaling Technology), AKT (1:2000 dilution, ab32505, Abcam), p-p53 (1:2000 dilution, s15, 9284, Cell Signaling Technology), p-JNK (1:2000 dilution, 9251, Cell Signaling Technology), JNK (1:2000 dilution, 9252, Cell Signaling Technology), c-Myc (1:2000 dilution, ab32072, Abcam), p53 (1:1000 dilution, sc-126, Santa Cruz), and Flag (1:5000 dilution, F1804, Sigma), mTOR (1:2000 dilution, 2983, Cell Signaling Technology), Rictor (1:2000 dilution, 2114, Cell Signaling Technology), TBK1 (1:2000 dilution, 3504, Cell Signaling Technology), IKBKE (1:2000 dilution, 3416, Cell Signaling Technology). β-Actin (1:5000 dilution, 66009-1-Ig, Proteintech) antibody was used as a loading control. Samples derived from the same experiment and gels/blots were processed in parallel.

Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay reagent (Bio-Rad) according to manufacturer instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-p21 (1:500; sc-397), anti-IκBα (1:500; sc-371), anti-p53 (1:500; sc-126, all from Santa Cruz Biotechnology), anti-p-p53 (1:500; 9286, Cell Signaling Technology), anti-ID1 (1:2,000; BCH-1-195-14, Biocheck, Foster City, CA, USA), anti-ID2 (1:500, sc-489, Santa Cruz Biotechnology), anti-ID3 (1:500, sc-490, Santa Cruz Biotechnology), anti-ID4 (1:200; ab49261, Abcam), and anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with a horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).

The lysis buffer was utilized to extract total proteins from C-28/I2 chondrocytes and the isolated proteins were quantified with a BCA kit (#71,285-M, Sigma-Aldrich, USA), followed by loading 30 μg proteins of each sample onto the 12% SDS-PAGE. After resolving for 1 hour, proteins in the gel were further transferred onto the PVDF membrane (#1,620,256 Bio-Rad, USA), and incubated with 5% BSA. Then, the membrane was incubated with the primary antibodies against p-ATM (1:800, # sc-47,739 Santa Cruz Biotechnology, Texas, USA), ATM (1:3000, # sc-135,663 Santa Cruz Biotechnology, Texas, USA), p-CHK2 (1:800, #ab278548 Abcam, Cambridge, UK), CHK2 (1:2000, #sc-17,747 Santa Cruz Biotechnology, Texas, USA), p21 (1:1000, #sc-6246 Santa Cruz Biotechnology, Texas, USA), p53 (1:2000, #sc-126 Santa Cruz Biotechnology, Texas, USA), and β-actin (1:5000, #sc-47,778 Santa Cruz Biotechnology, Texas, USA). Then the secondary antibody (1:2000, #sc-2359 Santa Cruz Biotechnology, Texas, USA) was utilized to incubate with the membrane for 1.5 h under room temperature. Lastly, the bands were incubated with the ECL solution, followed by quantification using the Image J software [21 (link)].

Cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail (11,836,153,001, Complete Mini, Roche, Mannheim, Germany) and phosphatase inhibitor cocktail (04906845001, PhosSTOP EASYpack, Roche). Cell debris were then removed by centrifugation at 4 °C. After centrifugation, protein concentrations were measured with Pierce BCA Protein Assay Kit (23,225, Thermo Fisher Scientific, Rockford, IL, USA). For western blotting, 30 μg of protein lysates from each established cell line were separated by 8~10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto nitrocellulose membranes (IPVH00010, Millipore, Bedford, MA, USA), blocked at room temperature for 1 h with 5% skim milk, and incubated at 4 °C overnight with the following primary antibodies: p53 (1:1000, sc-126, Santa Cruz Biotechnology, CA, USA), β-actin (1:1000, sc-47,778, Santa Cruz Biotechnology), and EGFP (1:1000, ab184601, Abcam, Cambridge, UK). After three 15-min washes with Tris-buffered saline containing 0.1% Tween 20, the blots were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody: goat anti-Mouse IgG(H+L)-HRP (1:4000, #SA001–500, GenDEPOT, Barker, TX, USA). Proteins were detected using chemiluminescent reagent, ECL solution (#W6002, Biosesang, Gyeonggi, Korea).