Figure 5. Effect of protein kinase inhibitors on phosphorylation of LRP-1. Retinal ganglion cell (RGC)-5 cells were left untreated or treated for 24 h with staurosporine (SS; 2.0 μM), norrin (25 ng/ml), with or without H-89 (n=3 experiments). At the end of 24 h, proteins were extracted and immunoprecipitated by using antibodies against lipoprotein-related receptor-1 (LRP-1). Immunoprecipitated proteins were then subjected to western blot analysis by using antibodies against phosphoserine, phosphotyrosine, and LRP-1 (A). Relative amount of proteins was determined by densitometric analysis (B). LRP-1 was expressed constitutively in norrin-treated cells and its expression did not change regardless of treatment condition (A). LRP-1 was phosphorylated at Tyr-residues constitutively in norrin-treated cells and phosphorylation status of LRP-1 at Tyr-residues did not change with any of the treatment condition (A). In addition, LPR-1 was constitutively phosphorylated at Ser-residues in norrin-treated cells (A). Compared to Ser-phosphorylation of LRP-1 in norrin-treated cells, Ser-phosphorylation of LRP-1 was significantly reduced when cells were treated with H-89 (A, B, *p<0.05). In addition, compared to Ser-phosphorylation of LRP-1 under SS and norrin-treated conditions, Ser-phosphorylation of LRP-1 was further reduced under H-89, SS, and norrin-treated conditions (A,B, **p<0.05). Cell viability assays indicate that, in the absence of SS, lower concentrations of H-89 had no effect on cell survival, but higher concentrations of H-89 (2.5–10.0 μM) decreased cell survival significantly regardless of norrin's presence (C, *p<0.05). Furthermore, in the presence of SS, lower concentrations of H-89 had no effect on cell survival, but higher concentrations of H-89 (2.5–10.0 μM) decreased norrin-mediated cell survival (D, *p<0.05).