Western Blot Analysis of Signaling Proteins

Western blot analysis was performed as described previously [33 (link)] with minor modifications. Briefly, aliquots of 30–60 µg of protein were subjected to 8–12% SDS-PAGE and transferred onto Hybond-P⁺ polyvinylidene difluoride membranes (Amersham, Buckinghamshire, UK). The membranes were incubated with the following primary antibodies: anti-ICAM-1 (ab225884, 1:1000, Abcam, Cambridge, UK), anti-VCAM-1 (ab106778, 1:1000, Abcam), anti-phospho-ERK (sc-7383, 1:1000, Santa Cruz Biotechnology), anti-total-ERK (sc-94 1:1000, Santa Cruz Biotechnology), anti-phospho-PKC (9375S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-PKC (sc-10800, 1:1000, Santa Cruz Biotechnology), anti-phospho-PDK1 (3061S, 1:1000, Cell Signaling Technology), anti-PDK1 (3062S, 1:1000, Cell Signaling Technology), anti-phospho-STAT-3 (9131S, 1:1000, Cell Signaling Technology, anti-STAT-3 (4904S, 1:1000, Cell Signaling Technology), anti-HIF-1α (ab2185, 1:1000, Abcam), anti-phospho-NF-κB (8242S, 1:1000, Cell Signaling Technology), anti-NF-κB (8242S 1:1000, Cell Signaling Technology), and anti-β-actin (A2066, 1:2000, Sigma-Aldrich, St. Louis, MO, USA). The bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and an ECL western blotting detection reagent (Bio-Rad, Hercules, CA, USA).