2.7. Western Blot for Intracellular Sox5, Sox6, Sox9, and ERK in Isolated Chondrocytes and Cartilage Explants

For chondrocytes, each well was washed with ice-cold phosphate-buffered saline, then lysed with 250 μl of ice-cold radioimmunoprecipitation assay (RIPA) buffer following a standard protocol (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% (volume/volume) Triton X-100, 0.1% (weight/volume) SDS, 1% (w/v) sodium deoxycholate, 0.5% (v/v) Igepal CA-630, 1 mM phenylmethylsulfonyl fluoride, 10 μM E64, 1 μg/ml pepstatin A, and 10 μg/ml aprotinin) [13]. Lysates were removed after 45 min and centrifuged (14,000 x g, 15 min). Protein concentration was determined by the Bradford colorimetric procedure (Bio-Rad) [14]. 15 μg of protein was mixed with 4x sample buffer, boiled (5 min) and electrophoresed (precast 4-12% bis-tris gradient, Invitrogen NuPAGE (Thermo Fisher Scientific), UK), transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) and blocked (30 min) in 5% dried milk. Membranes were treated with either rabbit-polyclonal anti-human Sox5, Sox6, or Sox9 primary antibody for 1 h, washed 3 times (PBS+0.05% Tween 20), and then incubated for 1 h with HRP antibody. Antibody staining was visualised with enhanced chemiluminescence (ECL). Expression of multiple proteins, including the loading control ERK, was achieved by stripping antibodies with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) for 30 min at 50°C with stirring, and reprobing.

For cartilage explants, 1 g of cartilage tissue was dissected into 2 ml of ice-cold 15 mM HEPES buffer and snap frozen in liquid nitrogen for 5 min and thawed at 37°C 3 times. Cellular protein was extracted using RIPA at 4°C for 2 h with agitation. Extracts were centrifuged (13,000 x g, 20 min). Protein concentration was determined, and samples were prepared and analysed as above.