Western Blot Analysis of Visfatin Signaling

For Western blotting, JJ012 and SW1353 cells (5 × 10⁵ each) were seeded in six-well plates. After 24 h of treatment with various concentrations of visfatin, the cells were collected and lysed. A total of 50 μg of protein per lane was separated on a 10% SDS-PAGE gel, transferred to a membrane, and probed with primary antibodies, including p-RAF (SC-101791, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-MEK (2338S, Cell Signaling, Danvers, MA, USA), p-ERK (SC-7383, Santa Cruz Biotechnology, Santa Cruz, CA, USA), HIF-1α (ab1, Abcam, Cambridge, UK), RAF (SC-133, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MEK (SC-6250, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ERK (SC-1647, Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGF-D (A19242, ABclonal, Woburn, MA, USA), and β-Actin (A5441, Sigma, St. Louis, MO, USA). Appropriate horseradish peroxidase-conjugated secondary antibodies were applied in TBS containing 5% non-fat milk. Protein bands were detected on the blots using enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Glasgow, UK). The data were expressed as a relative expression ratio to β-Actin [37 (link)].