Hsp90α

Western blot analysis was performed as previously described 35 (link). Briefly, cells lysed in RIPA lysis buffer (Beyotime, China), after being washed in cold PBS, were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat dry milk in TBST buffer (10 mM Tris-HCl, 100 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature before incubation with HSP90α (Abcam, mouse mAb; 1:1000) and β-actin (Abcam, mouse mAb; 1:1000) primary antibodies. After being washed to remove non-bound primary antibodies, the membranes were incubated with the corresponding secondary antibody at a 1:2000-10000 dilution at room temperature for 2 h. The membranes were washed three times with TBST, and immunoreactive signals were detected using western blot Luminol Reagent (Beyotime, China), according to the manufacturer's instructions.

Equal amounts of protein samples were separated using the SDS-PAGE technique and afterward transferred onto nitrocellulose membranes. Ponceau S staining was used for electro-transfer uniformity and normalization purposes. Thereafter, the membranes were washed and blocked with 2% BSA in TRIS-buffered saline containing 0.05% Tween 20, pH 7.6, and exposed for 2 h to the primary calreticulin (Thermo Scientific, catalog # PA3-900), HSP 90-α (Abcam, Cambridge, UK, catalog # ab13492), HSP 60 (Thermo Scientific, catalog # MA3-013), HSPB1 (Abcam, catalog # ab79868), and Annexin A1 (Thermo Scientific, catalog # PA5-22266) antibodies in TBS with 1% BSA followed by the appropriate IgG coupled with horse radish peroxidase (IgG–HRP) secondary antibodies for 1 h (Abcam, catalog # ab6721, and Sigma-Aldrich, catalog # A2304). The subsequent chemiluminescence reaction was revealed using the ECL Western Blotting Substrate kit (Thermo Scientific) and images were taken with the Image Quant LAS 4000 camera system (GE Healthcare, Uppsala, Sweden). Thereafter, Digital densitometry analysis was performed using the ImageJ analysis freeware. All original, unaltered, and unprocessed membrane and Ponceau S-colored membrane images can be found in Figures S1–S6 as Supplementary Material.

MSCs were treated with 1000 ng/mL cisplatin for 4 hours, and cells were harvested 12 and 24 hours later. Each sample containing 10 μg of total protein from whole-cell lysates was run on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Membranes were probed with primary antibodies against HSP90α (Abcam, Cambridge, UK), HSP90β (Abcam), HSP72 (LifeSpan Biosciences, Eching, Germany), HSP27 (Cell Signaling Technology, Leiden, Netherlands), HSP60 (Cell Signaling Technology), and HSP10 (Santa Cruz, Heidelberg, Germany). β-actin was used as a loading control. Blots were visualized on X-ray film using a horseradish-peroxidase kit (Cell Signaling Technology).