Figure 6
Figure 6. Role of Axl/Tyro3/Mer RTK in the activity of human protein S. (A) The mRNA levels of Axl, Tyro3, and Mer RTK in THP-1 monocytes and THP-1 macrophages were analyzed by real-time PCR. Results were normalized to the amount of GAPDH mRNA. (B) Mer protein expression. THP-1 macrophages were treated with protein S for 24 hours. Protein levels of Mer were detected by Western blot analysis. β-Actin was used as an internal control. (C) Ligand binding assay. Purified human protein S (10 nM) was mixed with 10 nM Mer/Fc fusion protein or tropomyosin-related kinase B (TrkB)/Fc as a negative control at 4°C for 2 hours, followed by incubation with protein A agarose beads for additional 1 hour. Bound proteins were subjected to SDS-PAGE and immunoblotted with anti–protein S antibody. Protein S input (20%). (D) Tyrosine phosphorylation assay. THP-1 macrophages were serum-starved for 6 hours prior to the human protein S treatment (20 μg/mL) for 30 minutes at 37°C. The cell lysates were immunoprecipitated with goat anti-Mer antibody, and immunoblotted with a phosphotyrosine-specific monoclonal antibody or anti-Mer. (E) Herbimycin A (a broad-spectrum RTK inhibitor) blocking. THP-1 macrophages were treated with protein S plus 20 ng/mL herbimycin A for 24 hours, followed by incubation with 5 μg/mL Alexa Fluor 488–AcLDL in the presence or absence of 250 μg/mL AcLDL for 3 hours at 37°C. (F) Mer/Fc chimera blocking the effects of protein S on AcLDL uptake. THP-1 macrophages were treated with protein S plus receptor/Fc chimera (Axl/Fc, Tyr3/Fc, or Mer/Fc) for 24 hours, followed by incubation with 5 μg/mL Alexa Fluor 488–AcLDL in the presence or absence of 250 μg/mL AcLDL for 3 hours at 37°C. (G) Mer/Fc chimera blocking the effects of protein S on SR-A protein expression. THP-1 macrophages were treated with protein S with or without Mer/Fc chimera for 24 hours. The SR-A protein levels were detected by Western blot analysis. β-Actin was used as an internal control. (H) Mer/Fc chimera blocking the effects of protein S on SR-A mRNA expression. THP-1 macrophages were treated with protein S with or without Mer/Fc chimera for 24 hours. The SR-A mRNA levels were detected by real-time PCR. Results were normalized to the amount of GAPDH mRNA. Data represent mean plus or minus SD of 3 experiments. *P < .05 versus untreated controls; #P < .05 versus protein S–treated group.

Role of Axl/Tyro3/Mer RTK in the activity of human protein S. (A) The mRNA levels of Axl, Tyro3, and Mer RTK in THP-1 monocytes and THP-1 macrophages were analyzed by real-time PCR. Results were normalized to the amount of GAPDH mRNA. (B) Mer protein expression. THP-1 macrophages were treated with protein S for 24 hours. Protein levels of Mer were detected by Western blot analysis. β-Actin was used as an internal control. (C) Ligand binding assay. Purified human protein S (10 nM) was mixed with 10 nM Mer/Fc fusion protein or tropomyosin-related kinase B (TrkB)/Fc as a negative control at 4°C for 2 hours, followed by incubation with protein A agarose beads for additional 1 hour. Bound proteins were subjected to SDS-PAGE and immunoblotted with anti–protein S antibody. Protein S input (20%). (D) Tyrosine phosphorylation assay. THP-1 macrophages were serum-starved for 6 hours prior to the human protein S treatment (20 μg/mL) for 30 minutes at 37°C. The cell lysates were immunoprecipitated with goat anti-Mer antibody, and immunoblotted with a phosphotyrosine-specific monoclonal antibody or anti-Mer. (E) Herbimycin A (a broad-spectrum RTK inhibitor) blocking. THP-1 macrophages were treated with protein S plus 20 ng/mL herbimycin A for 24 hours, followed by incubation with 5 μg/mL Alexa Fluor 488–AcLDL in the presence or absence of 250 μg/mL AcLDL for 3 hours at 37°C. (F) Mer/Fc chimera blocking the effects of protein S on AcLDL uptake. THP-1 macrophages were treated with protein S plus receptor/Fc chimera (Axl/Fc, Tyr3/Fc, or Mer/Fc) for 24 hours, followed by incubation with 5 μg/mL Alexa Fluor 488–AcLDL in the presence or absence of 250 μg/mL AcLDL for 3 hours at 37°C. (G) Mer/Fc chimera blocking the effects of protein S on SR-A protein expression. THP-1 macrophages were treated with protein S with or without Mer/Fc chimera for 24 hours. The SR-A protein levels were detected by Western blot analysis. β-Actin was used as an internal control. (H) Mer/Fc chimera blocking the effects of protein S on SR-A mRNA expression. THP-1 macrophages were treated with protein S with or without Mer/Fc chimera for 24 hours. The SR-A mRNA levels were detected by real-time PCR. Results were normalized to the amount of GAPDH mRNA. Data represent mean plus or minus SD of 3 experiments. *P < .05 versus untreated controls; #P < .05 versus protein S–treated group.

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