“…25 Briefly, 10-μg protein samples were resuspended in a reduced sample buffer, and then electrophoresed on a 4-12% Bis-Tris gel (Novex, San Diego, CA, USA) with MOPS running buffer, blotted to nitrocellulose, and sequentially probed with polyclonal rabbit antisera raised against Hsp90, Hsp70, Hsp60, Hsp32, Hsp27, Akt, phosphorylated-Akt (pAkt), eNOS, phosphorylated-eNOS (p-eNOS) or actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). A horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was then added, and secondary antibodies were detected through autoradiography using enhanced chemiluminescence (GE Healthcare UK Ltd, Little Chalfont, UK).…”