Figure 7.
Figure 7. GMFG is associated with the mitochondrial membrane protein ATAD3A. (A) Cellular lysates of human THP-1 cells transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours were immunoprecipitated with control immunoglobulin G (IgG) or monoclonal anti-GMFG antibodies. The immunoprecipitated proteins were isolated using Dynabeads protein G, eluted, and subjected to liquid chromatography-tandem mass spectrometry analysis. Proteome data from Ctrl siRNA- or GMFG siRNA-transfected THP-1 cells were analyzed by MaxQuant, including protein name, intensity L (Ctrl siRNA- or GMFG siRNA-transfected cells immunoprecipitated with IgG antibody), intensity H (Ctrl siRNA-transfected cells immunoprecipitated with GMFG antibody), intensity M (GMFG siRNA-transfected cells immunoprecipitated with GMFG antibody), normalized H/L ratio, H/M ratio, and M/L ratio. (B-C) Immunoprecipitation analysis of GMFG association with the mitochondrial membrane protein ATAD3A in human THP-1 cells. Total cellular lysates were immunoprecipitated with anti-GMFG antibody (B) or anti-ATAD3A antibody (C), then the immunoprecipitants subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates) are shown in the left 2 lanes; immunoprecipitates (IPs) are shown in the right 2 lanes. (D) Cellular lysates of human HEK-293T cells cotransfected with GFP-tagged GMFG plasmid and Myc-DDK–tagged ATAD3A plasmid for 48 hours were immunoprecipitated with control IgG, anti-GFP, or anti-Myc antibody; the IPs were then subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates; bottom). Each sample corresponds to 5% of the cell lysate used in each immunoprecipitation. (E) Immunoblot analysis of ATAD3A proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control.

GMFG is associated with the mitochondrial membrane protein ATAD3A. (A) Cellular lysates of human THP-1 cells transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours were immunoprecipitated with control immunoglobulin G (IgG) or monoclonal anti-GMFG antibodies. The immunoprecipitated proteins were isolated using Dynabeads protein G, eluted, and subjected to liquid chromatography-tandem mass spectrometry analysis. Proteome data from Ctrl siRNA- or GMFG siRNA-transfected THP-1 cells were analyzed by MaxQuant, including protein name, intensity L (Ctrl siRNA- or GMFG siRNA-transfected cells immunoprecipitated with IgG antibody), intensity H (Ctrl siRNA-transfected cells immunoprecipitated with GMFG antibody), intensity M (GMFG siRNA-transfected cells immunoprecipitated with GMFG antibody), normalized H/L ratio, H/M ratio, and M/L ratio. (B-C) Immunoprecipitation analysis of GMFG association with the mitochondrial membrane protein ATAD3A in human THP-1 cells. Total cellular lysates were immunoprecipitated with anti-GMFG antibody (B) or anti-ATAD3A antibody (C), then the immunoprecipitants subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates) are shown in the left 2 lanes; immunoprecipitates (IPs) are shown in the right 2 lanes. (D) Cellular lysates of human HEK-293T cells cotransfected with GFP-tagged GMFG plasmid and Myc-DDK–tagged ATAD3A plasmid for 48 hours were immunoprecipitated with control IgG, anti-GFP, or anti-Myc antibody; the IPs were then subjected to immunoblot analysis with anti-ATAD3A or anti-GMFG antibody. Samples of the total lysate after immunoprecipitated complexes/beads were isolated (lysates; bottom). Each sample corresponds to 5% of the cell lysate used in each immunoprecipitation. (E) Immunoblot analysis of ATAD3A proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. α-tubulin was used as a loading control.

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