Hsp70

Total protein was extracted from cells using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher, Houston, TX, USA) containing 50 mM Tris–HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, and 1% Triton X, plus Halt protease and phosphatase inhibitor (Thermo Fisher). Protein was quantified using Pierce BCA protein assay (Thermo Scientific) and read on a BioTek microplate reader at 570 nm. Protein was denatured by heating at 95 °C for 5 min and separated on a gradient SDS polyacrylamide gel electrophoresis gel (Thermo Fisher). The proteins were transferred onto a nitrocellulose membrane (LI-COR #926-31092, Lincoln, NE, USA), blocked using Odyssey blocking buffer (LI-COR #927-40000), incubated overnight with antibodies as follows, and visualized using a LI-COR Odyssey fluorescence scanner. The primary antibodies were Nrf2 (SC-722) 1:200 rabbit polyclonal (Santa Cruz Biotechnologies), GAPDH (MAB374) 1:1,000 mouse monoclonal (Chemicon Int., Temecula, CA, USA), HSP70 (SC 33575) 1:1,000 rabbit polyclonal (Santa Cruz Biotechnologies), tumor necrosis factor receptor–associated factor 6 (TRAF6; 04-451) 1:1,000 rabbit monoclonal (Chemicon), and HMOX-1 (SC10789) 1:100 rabbit polyclonal (Santa Cruz Biotechnologies), as shown in Table 2.

Western blotting was performed as described previously.4 Samples of equivalent total protein (20 μg) were loaded. Primary antibodies against CD63, CD81, HSP70 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β‐Actin, ZIP4, Adiponectin, MMP9 and MMP12 (ProteinTech Group, Rosemont, IL, USA) were used.