A well-characterized Müller cell line, MIO-M1, was used in our studies.
24 Cells were grown in Gibco Dulbecco's modified Eagle's medium (containing 4500 mg/L glucose, sodium pyruvate and stabilized
l-glutamine [GlutaMAX; Invitrogen, Paisley, UK]) plus 10% vol/vol fetal bovine serum and penicillin/streptomycin (Invitrogen), at 37°C with 5% CO
2. Experiments designed to investigate the LRP1 expression in mouse retinas were performed on Müller glial cell cultures obtained using a method previously described.
25 Mouse J774A.1 macrophage-derived cells
16,26 and the rat JHU-4 prostate (MAT-Lu) cell line (this article) were used as positive controls of LRP1 expression, whereas Chinese hamster ovary (CHOK-1) 13–5-1 was used as the LRP1-deficient cell line.
27 α
2M was purified from human plasma following a procedure previously reported,
28 and the activated form of α
2M (α
2M*) was generated by incubating α
2M with 200 mM methylamine-HCl for 6 hours at pH 8.2, as previously described.
29 An expression construct, encoding RAP as a glutathione
S-transferase (GST) fusion protein (GST-RAP), was provided by Guojum Bu (Washington University, St. Louis, MO). GST-RAP was expressed and purified as reported previously
30 and was used without further modification. Immunoblots were performed with the following primary antibodies: polyclonal rabbit anti-GFAP (Dako, Carpinteria, CA), mouse monoclonal anti–phosphorylated ERK1/2 (anti–p-ERK1/2), polyclonal rabbit anti–total ERK1/2, monoclonal anti–phosphorylated STAT3 (anti–p-STAT3), anti–calreticulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and mouse monoclonal antibody anti–β-subunit LRP1 (clone 5A6), which was kindly provided by Dudley Strickland (University of Maryland School of Medicine, Baltimore, MD). Immunocytochemical studies were performed with the same GFAP and LRP1 antibodies, and additional studies were carried out using a monoclonal mouse anti–vimentin (Dako) and a polyclonal rabbit anti–cellular retinaldehyde-binding protein (CRALBP) kindly donated by John C. Saari (University of Washington, Seattle, WA).