polyclonal antibodies against lipoprotein-related receptor-1 (lrp-1)  (Orbigen Inc)

Journal: Molecular Vision

Article Title: Norrin attenuates protease-mediated death of transformed retinal ganglion cells

doi:

Figure Lengend Snippet: 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.

Article Snippet: Aliquots containing 500 μg total proteins from each treatment were incubated overnight 4 °C with 2 μl polyclonal antibodies against lipoprotein-related receptor-1 (LRP-1; Orbigen, San Diego, CA).

Techniques: Immunoprecipitation, Western Blot, Activity Assay, Zymography, Expressing

Journal: Molecular Vision

Article Title: Norrin attenuates protease-mediated death of transformed retinal ganglion cells

doi:

Figure Lengend Snippet: 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).

Article Snippet: Aliquots containing 500 μg total proteins from each treatment were incubated overnight 4 °C with 2 μl polyclonal antibodies against lipoprotein-related receptor-1 (LRP-1; Orbigen, San Diego, CA).

Techniques: Immunoprecipitation, Western Blot, Expressing