Sodium dodecyl sulphate (sds)

Total RNA was prepared from cultured tenocytes, chondrocytes, NIH3T3 cells, C3H10T1/2 cells, ATDC5 cells, and MC3T3-E1 cells as described previously^{25 (link)}. Total RNA (15 μg) was denatured with 6% formaldehyde, electrophoresed on a 1% agarose gel, and transferred onto Nytran membranes with Turboblotter (Schleicher and Schuell, Whatman plc, Maidstone, UK). Rat Tnmd and rat Scx probes were amplified from cDNA prepared from cultured rat tenocytes with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Probes for mouse Tnmd, mouse Scx, and rat type II collagen alpha 1 (Col2a1) were described previously^{10 ,25 (link),36 (link)}. A probe for rat type I collagen alpha 2 (Col1a2) was generously provided by Dr. Bjorn Olsen. A probe for mouse Twist1 was amplified from a cDNA clone purchased from Open Biosystems (Lafayette, CO, USA). Hybridisation was performed overnight at 42 °C with an appropriate cDNA probe labelled with [α-³²P]dCTP in a solution containing 50% formamide (Wako, Osaka, Japan), 6 X saline-sodium phosphate-EDTA buffer (Sigma-Aldrich), 0.1% bovine serum albumin (Sigma-Aldrich), 0.1% Ficoll 400 (GE-Healthcare), 0.1% polyvinylpyrrolidone (Wako), 0.5% sodium dodecyl sulphate (Wako), and 100 μg/mL denatured salmon sperm DNA (Wako).

The other halves of the tape strips were also further cut into 1/8 pieces. All pieces were immersed in 1 mL alkaline aqueous solution [0.1 N NaOH (Wako, Osaka, Japan), 1% (w/v) sodium dodecyl sulphate (Wako, Osaka, Japan)] and then incubated at 60 °C for 2.5 h. After incubation, the solution was neutralized with 1 N HCl (Wako, Osaka, Japan). The total protein content in each sample solution was measured by a BCA kit (Thermo Scientific, Rockford, IL, USA) using bovine serum albumin as a protein standard. The AMP concentrations measured using the TS method were corrected with the total protein content.

Whole cell lysates were extracted using RIPA buffer containing a proteinase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and PhosSTOP (Roche, Basel, Switzerland). Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples (25 µg) were loaded on NuPAGE 4–12% Bis–Tris gels (Thermo Fisher Scientific) and transferred to polyvinylidene fluoride membranes (GE Healthcare, Chicago, IL, US). Thereafter, membranes were blocked with 5% bovine serum albumin (Sigma) or 5% skim milk (Wako) in TBS with Tween-20 (TBST; Takara Bio) for 1 h. Next, membranes were incubated with the primary antibodies listed in Table 2 at 4 °C for 24 h. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (1:5000; GE Healthcare) at RT for 1 h, and then detected with ECL Select Western Blotting Detection Reagent (GE Healthcare). Protein bands were visualised using a Molecular Imager ChemiDoc XRS + system (Bio-Rad Laboratories, Hercules, CA, USA). For stripping the protein bands, membranes were incubated at 50 °C for 30 min in stripping buffer, which was composed of 10% sodium dodecyl sulphate (Wako), 0.5 M tris (hydroxymethyl) aminomethane hydrochloride (Nacalai Tesque), and 100 mM 2-Mercaptoethanol (Sigma). Membranes were then blocked and incubated with subsequent antibodies.