Figure 3
Figure 3. HGAL interacts with actin and myosin II proteins, and the interaction with the myosin is increased by IL-6–induced HGAL phosphorylation. (A) Cellular lysates of unstimulated HeLa-HGAL and SUDHL6 cells were immunoprecipitated with either anti-HGAL or anti-actin antibodies and blotted with the indicated antibodies. Unconjugated beads served as a control (“B”). The size of HGAL in the HeLa cells is 26 kDa, since the cloned protein is a fusion protein with V5 tag. (B) Cellular lysates were extracted from unstimulated and IL-6–stimulated SUDHL6 cells, immunoprecipitated with anti-HGAL antibody, resolved by SDS-PAGE, and stained with Coomassie blue. Protein bands of 250 kDa, 230 kDa, and 140 kDa (→) were excised and analyzed by mass spectrometry analysis and sequencing that revealed that all the 3 proteins represent myosin II, the expected size of which is 250 kDa. (C) Cellular lysates were extracted from unstimulated SUDHL6 and VAL lymphoma cell lines, immunoprecipitated with anti-HGAL antibody, and blotted with indicated antibodies. Unconjugated beads served as a control (“B”). (D) Cellular lysates were extracted from unstimulated or IL-6–stimulated SUDHL6 cells and immunoprecipitated with either anti-HGAL or antimyosin antibodies, resolved on the SDS-PAGE, and blotted with the indicated antibodies. (E) HeLa-HGAL cells were treated with cytochalasin D (5 μM) and latrunculin B (5 μM) for 30 minutes, respectively, to inhibit actin polymerization and disrupt actin filaments. The whole-cell lysates were extracted, immunoprecipitated with anti-HGAL antibody, and blotted with the indicated antibodies. (F) SUDHL6 and HeLa-HGAL cells were stimulated with IL-6 for up to 60 minutes. At the indicated time points, cellular lysates were extracted, immunoprecipitated with anti-HGAL antibody, and blotted with the indicated antibodies. Arrows indicate immunoglobulin light chains. (G) Unstimulated or IL-6–stimulated Hela-HGAL (left) and SUDHL6 (right) cells were fixed with 4% paraformaldehyde, stained with anti-HGAL antibody (green), antimyosin antibody (red), and DAPI (blue), and visualized with the Carl Zeiss LSM510 microscope. (H) HGAL and myosin II colocalization in podosome-like structures of IL-6–stimulated SUDHL6 cells, as visualized with the confocal Carl Zeiss LSM510 microscope (see “Immunofluorescence microscopy” for detailed image acquisition information). Colors as described in panel G. In panels A,C-E, representative blots of 3 independent experiments are shown.

HGAL interacts with actin and myosin II proteins, and the interaction with the myosin is increased by IL-6–induced HGAL phosphorylation. (A) Cellular lysates of unstimulated HeLa-HGAL and SUDHL6 cells were immunoprecipitated with either anti-HGAL or anti-actin antibodies and blotted with the indicated antibodies. Unconjugated beads served as a control (“B”). The size of HGAL in the HeLa cells is 26 kDa, since the cloned protein is a fusion protein with V5 tag. (B) Cellular lysates were extracted from unstimulated and IL-6–stimulated SUDHL6 cells, immunoprecipitated with anti-HGAL antibody, resolved by SDS-PAGE, and stained with Coomassie blue. Protein bands of 250 kDa, 230 kDa, and 140 kDa (→) were excised and analyzed by mass spectrometry analysis and sequencing that revealed that all the 3 proteins represent myosin II, the expected size of which is 250 kDa. (C) Cellular lysates were extracted from unstimulated SUDHL6 and VAL lymphoma cell lines, immunoprecipitated with anti-HGAL antibody, and blotted with indicated antibodies. Unconjugated beads served as a control (“B”). (D) Cellular lysates were extracted from unstimulated or IL-6–stimulated SUDHL6 cells and immunoprecipitated with either anti-HGAL or antimyosin antibodies, resolved on the SDS-PAGE, and blotted with the indicated antibodies. (E) HeLa-HGAL cells were treated with cytochalasin D (5 μM) and latrunculin B (5 μM) for 30 minutes, respectively, to inhibit actin polymerization and disrupt actin filaments. The whole-cell lysates were extracted, immunoprecipitated with anti-HGAL antibody, and blotted with the indicated antibodies. (F) SUDHL6 and HeLa-HGAL cells were stimulated with IL-6 for up to 60 minutes. At the indicated time points, cellular lysates were extracted, immunoprecipitated with anti-HGAL antibody, and blotted with the indicated antibodies. Arrows indicate immunoglobulin light chains. (G) Unstimulated or IL-6–stimulated Hela-HGAL (left) and SUDHL6 (right) cells were fixed with 4% paraformaldehyde, stained with anti-HGAL antibody (green), antimyosin antibody (red), and DAPI (blue), and visualized with the Carl Zeiss LSM510 microscope. (H) HGAL and myosin II colocalization in podosome-like structures of IL-6–stimulated SUDHL6 cells, as visualized with the confocal Carl Zeiss LSM510 microscope (see “Immunofluorescence microscopy” for detailed image acquisition information). Colors as described in panel G. In panels A,C-E, representative blots of 3 independent experiments are shown.

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