Pia32963

Cells were lysed in a radio-immunoprecipitation assay buffer with protease inhibitors (PIA32963, Thermo Fisher Scientific) and phosphatase inhibitors (2,006,643, Calbiochem, Billerica, MA, USA). After cell lysis, proteins were fractionated by 10%–15% SDS gels and electro-transferred to polyvinylidene difluoride transfer membranes (IPVH00010, Millipore, Billerica, MA, USA). After blocking 1 h with a blocking buffer (1,706,404, Bio-Rad, Hercules, CA, USA), the membrane was incubated overnight with primary antibodies and then with secondary antibodies conjugated with horseradish peroxidase for 45 min (7074 S/7076 S, Cell Signaling, Danvers, MA, USA). We used antibodies against Lrp5, Runx 2, Snail, MSN, Calr, cleaved-caspase 3, caspase 3, CD91, p-CREB, and CREB (Cell Signaling), c-fos, NFATc1, Cathepsin K (Santa Cruz Biotechnology, Dallas, TX, USA), CD47 (Thermo Fisher Scientific), Collagen I (Novus Biologicals, CO, USA), Osteocalcin (abcam, Boston, MA, USA), and β-actin as a control (A5441, Sigma). The protein level was determined using a SuperSignal west femto maximum sensitivity substrate (PI34096, Thermo Fisher Scientific), and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities.⁴¹ (link) The levels of Calr and MSN in CW008-treated CM were determined using the ELISA kits (MBS263181 and MBS2709503; MyBioSource).

Using a RIPA buffer with protease and phosphatase inhibitors (PIA32963, ThermoFisher Scientific, and 2006643, Calbiochem, Billerica, MA, respectively), cells were lysed. SDS gels (10%–15%) were used to size-fractionate proteins, followed by an electrotransfer to transfer membranes (IPVH00010, Millipore, Billerica, MA). The membrane was blocked for 1 h with a blocking buffer (1706404, Bio-Rad, Hercules, CA) and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (7074/7076, Cell Signaling, Danvers, MA). Antibodies, used in this study were NFATc1, cathepsin K (Santa Cruz Biotechnology, Dallas, TX), Hsp90ab1 (Abcam, Cambridge, UK), Myh9 (3403S, Cell Signaling), and β-actin as a control (A5441, Sigma). A SuperSignal west femto maximum sensitivity substrate (PI34096, ThermoFisher Scientific) was used to visualize protein bands with a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan).⁴⁷ (link)