Figure 4. Effect of norrin on LRP-1 phosphorylation. Retinal ganglion cell (RGC)-5 cells were left untreated or treated for 24 h with 2.0 μM staurosporine (SS) alone or SS and 25 ng/ml norrin (n=2 experiments). At the end of 24 h, cells were collected, proteins were extracted, and immunoprecipitated by using antibodies against lipoprotein-related receptor-1 (LRP-1). Immunoprecipitated proteins were subjected to western blot analysis by using antibodies against anti-phosphoserine, anti-phosphotyrosine, and anti-LRP-1 (A). Relative levels of proteins were determined by densitometry (C). At the end of treatment, conditioned medium was collected, and proteolytic activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) was determined by zymography assays (B). Relative amount of proteolytic activity was determined by densitomety (D). LRP-1 was expressed constitutively in untreated cells and its expression did not change regardless of the treatment condition (A). LRP-1 was phosphorylated at Tyr-residues constitutively in untreated cells and phosphorylation status of LRP-1 at Tyr-residuces was not altered with any of the treatment conditions (A). LPR-1 was constitutively phosphorylated at Ser-residues in untreated cells (A). Norrin-treatment alone had no effect on LPR-1 phosphorylation at Ser-residues, but SS-treatment significantly reduced Ser-phosphorylation of LRP-1 (A, C, *p<0.05). Nonetheless, LPR-1 phosphorylation at Ser-residues was significantly increased in the presence of SS and norrin (A,C, **p<0.05). RGC-5 cells were left untreated or treated for 48 h with 2.0 μM SS and 25 ng/ml norrin in serum-free medium (n=3 experiments). Compared to low levels of uPA in untreated cells, levels of both uPA (B,D, *p<0.05) and tPA (B,D, %p<0.05) were increased in the presence of SS. In addition, compared to low levels of uPA in norrin-treated cells, levels of both uPA (B,D, **p<0.05) and tPA (B,D, %%p<0.05) were significantly increased in the presence of SS and norrin. Nonetheless, cell viability assays (E) indicate that survival of RGC-5 cells decreased significantly under SS-treated conditions (E,*p<0.05), but not under SS and norrin-treated conditions (E, **p<0.05). NS, not significant.