Sds sample loading buffer

Equal amounts of protein (20–30 µg depending on sample concentration and amount of protein of interest in the sample) were denatured at 98 °C for 10 min in a solution containing Bolt Sample Reducing Agent (ThermoFisher Scientific) and SDS Sample Loading Buffer (Sigma Aldrich), then fractionated on Bolt™ 4–12% Bis-Tris Plus Gels (ThermoFisher Scientific). Proteins were transferred to nitrocellulose membranes using the iBlot Gel Transfer Device (Invitrogen) and blocked with 3% bovine serum albumin (BSA) (Bovogen, VIC, Australia) in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. Membranes were incubated overnight, with primary antibodies (Table 1) diluted in blocking buffer. Membranes were incubated with the secondary antibodies conjugated to horseradish peroxidase (HRP) (Table 1) in blocking buffer for at least 1 h, then washed 3 times with TBST. Immunodetection and quantification were performed using the Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, IL, USA) and the Chemi-Doc™ Gel Imaging System (Bio-Rad, CA, USA). Band density was measured with Image Lab software (Bio-Rad) and normalised to the density of the housekeeping protein GAPDH.

hBMSC and OA-CH were washed two times with PBS and lysed with RIPA buffer (Thermo Scientific, Waltham, MA) containing phosphatase and proteinase inhibitors (Roche, Germany). The concentration of cellular protein and EVs was quantified using a BCA protein kit assay (see hBMSC EV Isolation). Cell lysates (10 ug) or EVs (10 ug) were mixed with SDS-sample loading buffer (#B7053, Sigma-Aldrich, Germany), followed by boiling for 5 min at 95°C, and subjected to 12% SDS-PAGE. The proteins were transferred to 0.22 μm PVDF membranes (Roche, Penzberg, Germany) after electrophoretic separation. Blot membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibodies on a shaker overnight at 4°C. After washing, the membranes were incubated with the appropriate horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology and Jackson ImmunoResearch, West Grove, PA). Proteins were examined using ECL detection reagents (Thermo Scientific; Germany), and signals from cell lysates were normalized to β-actin (except for EV band analysis). Antibodies used for this study are listed in Table 2.

The soluble nuclear extraction was diluted in 800 µL SubCell Lysis buffer-I (from Kit) supplemented with Protease Inhibitor Cocktail. 100 µL cytoplasmic fraction and diluted nuclear fraction were transferred to new microtubes, snap-frozen on dry ice and stored at −80 °C as the input samples. In the remaining cytoplasmic and nuclear fractions, 6 µL of monoclonal Anti-β-Actin antibody (Sigma, A2228, Taufkirchen, Germany) was added for each IP reaction and the microtubes were rotated in a shaker at 4 °C overnight. Then 30 µL Pierce Protein A/G Magnetic Beads (ThermoFisher Scientific, 88803, Waltham, USA) were added into each IP reaction and the microtubes were rotated in a shaker at 4 °C for 2 h. The microtubes were then placed on a magnetic separator to capture magnetic beads and the associated proteins. The supernatants were removed and the magnetic beads were washed 5 times with 1 mL washing buffer (20 mM Tris-HCl pH 7.4, 0.4 M NaCl, 1 mM EDTA, 0.5% Triton X-100, 10% glycerol, Roche Complete Protease Inhibitor Cocktail). The proteins precipitated were eluted in 150 µL 1× SDS Sample loading buffer (Sigma, S3401, Taufkirchen, Germany), and the input samples were thawed on ice and mixed with 100 µL 2× SDS Sample loading buffer. The protein samples were heated at 95 °C for 5 min and centrifuged at 2000×g for 1 min before gel loading.