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Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.
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Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.
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Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.
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Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.
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Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.

Journal: Cells

Article Title: Dissecting the Biological Functions of Various Isoforms of Ferredoxin Reductase for Cell Survival and DNA Damage Response

doi: 10.3390/cells15010062

Figure Lengend Snippet: Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.

Article Snippet: Rabbit polyclonal anti-FDXR antibody (catalog number: 15584-1-AP) and rabbit polyclonal anti-Flag antibody (catalog number: 20543-1-AP) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Expressing, Generated, Alternative Splicing, Binding Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Recombinant, Produced, Control, Immunofluorescence