Figure 3. The P39 peptide binds to a... | Rockefeller University Press

Figure 3.

$Figure 3. The P39 peptide binds to a macrophage-specific surface protein. (A) Primary Kupffer cells were incubated with heparinase I, chondroitinase ABC, or trypsin, as indicated. PBS buffer (no enzyme) served as a control. After enzyme treatment, 100 µg/ml biotinylated P39 peptide (green) and the anti-F4/80 antibody (red) were incubated for 1 h and visualized. The top row shows differential interference contrast (DIC) microscopy images merged with DAPI-stained nuclei (blue). P39 peptide binding was trypsin sensitive and unaffected by GAG-removing enzyme treatment. Bar, 20 µm. (B) Lysates from five different cell types were fractionated by gel electrophoresis, and binding of biotinylated P39 peptide to proteins on the blot was detected using alkaline phosphatase–conjugated streptavidin. KC, primary rat Kupffer cells; PtM, primary rat peritoneal macrophages; Raw, a mouse macrophage-like cell line; Hep, primary rat hepatocytes; ASM, primary rat airway smooth muscle cells. P39 peptide binding to an ∼110-kD protein band (arrows) was detected only for the macrophage-like cells (rat KC, rat PtM, and mouse Raw). (C) Kupffer cell fractions were tested for P39 peptide binding. M, membrane fraction; C, cytosolic fraction; I, insoluble fraction. P39 peptide bound only to an ∼110-kD membrane protein (arrow). (D) Activation of the human monocyte cell line THP-1 with PMA treatment for differentiation into a macrophage-like cell resulted in production of a protein recognized by the P39 peptide. No binding was detected for control THP-1 cells treated with DMSO carrier alone. An anti-actin antibody was used as a loading control. Immunofluorescence and far-Western blotting images are representative of two to three independent experiments.$

The P39 peptide binds to a macrophage-specific surface protein. (A) Primary Kupffer cells were incubated with heparinase I, chondroitinase ABC, or trypsin, as indicated. PBS buffer (no enzyme) served as a control. After enzyme treatment, 100 µg/ml biotinylated P39 peptide (green) and the anti-F4/80 antibody (red) were incubated for 1 h and visualized. The top row shows differential interference contrast (DIC) microscopy images merged with DAPI-stained nuclei (blue). P39 peptide binding was trypsin sensitive and unaffected by GAG-removing enzyme treatment. Bar, 20 µm. (B) Lysates from five different cell types were fractionated by gel electrophoresis, and binding of biotinylated P39 peptide to proteins on the blot was detected using alkaline phosphatase–conjugated streptavidin. KC, primary rat Kupffer cells; PtM, primary rat peritoneal macrophages; Raw, a mouse macrophage-like cell line; Hep, primary rat hepatocytes; ASM, primary rat airway smooth muscle cells. P39 peptide binding to an ∼110-kD protein band (arrows) was detected only for the macrophage-like cells (rat KC, rat PtM, and mouse Raw). (C) Kupffer cell fractions were tested for P39 peptide binding. M, membrane fraction; C, cytosolic fraction; I, insoluble fraction. P39 peptide bound only to an ∼110-kD membrane protein (arrow). (D) Activation of the human monocyte cell line THP-1 with PMA treatment for differentiation into a macrophage-like cell resulted in production of a protein recognized by the P39 peptide. No binding was detected for control THP-1 cells treated with DMSO carrier alone. An anti-actin antibody was used as a loading control. Immunofluorescence and far-Western blotting images are representative of two to three independent experiments.