Hsp90ab1

Cultured cells were lysed in a radio-immunoprecipitation assay buffer and proteins were fractionated by 10-15% SDS gels and electro-transferred to polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA). The membrane was incubated overnight with primary antibodies and then with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling, Danvers, MA, USA). Antibodies against c-Myc, Oct4, Sox2, Klf4, Lrp5, Runx2, Snail, TGFβ, Eno1, Eef2, vinculin, β-catenin, p-Akt, Akt, p-NFkB p65, NFkB p65, p-ERK, ERK (Cell Signaling), MMP9, NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA), TRAIL (Novus, Centennial, CO, USA), p53 (Invitrogen, Carlsbad, CA, USA), Hsp90ab1 (Abcam, Cambridge, UK), Kdm3a (Thermo Fisher Scientific) and β-actin as a control (Sigma) were employed. The level of proteins was determined using a SuperSignal west femto maximum sensitivity substrate (Thermo Fisher Scientific), and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities.

The expression levels of HSP90AB1, SMARCC1, p-Akt, BAX, BCL2, GCLM, GCLC, HO-1 (Abcam, Cambridge, UK), Caspase3 (CST, Massachusetts, USA), p-HSP90AB1, p-c-Fos, and p-c-Jun (ImmunoWay, Texas, USA) were detected. The ImageJ 7.0 software was used to analyze the bands.

Western blot analysis was conducted using the procedure previously described 46 (link). We used antibodies against Lrp5, Runx2, Snail, sclerostin, Calr, p-eIF2α, eIF2α, TGFβ, PDL1 (Cell Signaling, Danvers, MA, USA), LIF, Trail (Novus Biologicals, Centennial, CO, USA), MMP9, NFATc1, cathepsin K (Santa Cruz Biotechnology, Dallas, TX, USA), p53, CXCL2, Ppib (Invitrogen, Carlsbad, CA, USA), Hsp90ab1 (Abcam, Cambridge, UK), and β-actin (Sigma, Saint Louis, MO, USA). We also employed a proteome profiler mouse XL cytokine array kit (R&D Systems, Minneapolis, MN, USA) and determined the expression of 111 cytokines and chemokines in MSC-derived CM.