Dot blotter

Cell lysates of hOBs were prepared by using RIPA buffer, and micro Lowry assay was used to quantify total protein of hOBs. Cell lysates were diluted to obtain a protein content of 1 ng/ μL in ddH₂O. 20 ng protein was transferred to a nitrocellulose membrane via a Dot blotter (Carl Roth, Karlsruhe, Germany). Ponceau staining was performed to confirm the transfer of proteins. Membranes were washed with Tris-buffered saline with Tween 20 (TBS-T) to remove ponceau and then blocked with 5 % BSA in TBS-T for 1 h at ambient temperature. Membranes were incubated with primary antibodies (see detailed information in Table 2(Tab. 2)) overnight at + 4 °C, and GAPDH was used for the housekeeper. The next day membranes were incubated with the secondary antibodies for 2 h at ambient temperature after washing with TBS-T. ECL substrate solution was used for chemiluminescent signal development of membranes. Signals of proteins were detected by a ChemCam imager (INTAS, Göttingen, Germany), and the intensity of signals was quantified by using the ImageJ software (Weng et al., 2020[52 ]).

Protein levels in cell culture supernatants were determined by dot blot as previously described [22 ]. Cell culture supernatants (40 µL) were applied to a nitrocellulose membrane by a dot blotter (Carl Roth). All transferred proteins were visualized by a solution containing 0.1% w/v Ponceau S in 1% v/v acetic acid. The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline/Tween 20 (TBS-T; 10 mM Tris-HCl at pH 7.6, 0.15 mM NaCl, and 0.1% v/v Tween-20) at room temperature for 1 h. It was then incubated with anti-procollagen type I N-terminal propeptide (PINP; 1:1000 in TBS-T; Ref. abx131414, Abbexa, Cambridge, UK) and anti-osteonectin (1:1000 in TBS-T; Ref. sc-74295, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. The following day, the membrane was incubated with the corresponding secondary antibodies (1:10,000 in TBS-T; Santa Cruz Biotechnology) for 2 h after washing. The protein signal was detected by a charge-coupled device camera (INTAS Science Imaging, Göttingen, Germany) after addition of an enhanced chemiluminescence substrate solution. The signal intensity was quantified using the ImageJ software (NIH, Bethesda, MD, USA).