Western Blot Analysis of Cellular Proteins

The total cellular extracts of LECs were prepared in ice-cold radioimmune precipitation buffer (RIPA buffer), and Western analysis was carried out as described previously [7 (link),70 (link),71 (link),72 (link)]. The membranes were probed with anti-Nrf2 (sc-722, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Nrf2 (ab62352, Abcam^®, Cambridge, MA, USA), Anti-Klf9 (ab177158, Abcam^®, Cambridge, MA, USA), Anti-Klf9 (sc-12996, Santa Cruz Biotechnology, Dallas, TX, USA), Anti-Prdx6 antibody (LF-PA0011, Ab Frontier, South Korea), Tubulin (ab44928, Abcam^®, Cambridge, MA, USA) or β-actin (A2066, Sigma-Aldrich, St. Louis, MO, USA) or Lamin B1 (ab133741, Abcam^®, Cambridge, MA, USA) as an internal control to monitor levels of protein expressions. After secondary antibody (sc-2354 and sc-2768, Santa Cruz Biotechnology, Dallas, TX, USA) incubation, protein bands were visualized by incubating the membrane with luminol reagent (sc-2048; Santa Cruz Biotechnology, Dallas, TX, USA). Finally, images (bands) were recorded with a FUJIFILM-LAS-4000 luminescent image analyzer (FUJIFILM Medical Systems Inc., Hanover Park, IL, USA).