Human GFI-1 Antibody

Detection of GFI-1 in THP-1 Human Cell Line by Flow Cytometry.

THP-1 monocytic leukemia cell line was stained with Goat Anti-Human GFI-1SOX2 Affinity Purified Polyclonal Antibody (Catalog # AF3540, filled histogram) or control antibody (Catalog # AB-108-C, open histogram), followed by APC-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.

Detection of Human GFI-1 by Western Blot

GFI1 interacts with proteins involved in the DDR. a GFI1-Flag fusion protein was immunoprecipitated from 293T cells. Co-precipitated proteins were run on polyacrylamide gel and stained with Coomassie blue. b Peptide sequence of MRE11 with peptides identified through mass spectrometry highlighted and corresponding spectra below. c Peptide sequence of PRMT1 with peptides identified through mass spectrometry highlighted and corresponding spectra below. d Variants of the GFI1-Flag fusion protein were immunoprecipitated from 293T cells. Extracts were separated by SDS–PAGE and blotted for the indicated proteins. e Diagram of the different GFI1 fusion proteins used in d and g. f GFI1-Flag fusion protein was immunoprecipitated in 293T cells in the presence or absence of benzonase. Extracts were separated by SDS–PAGE and blotted for MRE11. g GFI1 KO Jurkat cells were electroporated with plasmids expressing GFI1 variant constructs as shown in e. GFI1 KO and parental control Jurkat cells were used as controls. After 24 h, cells were exposed to 5 Gy IR and allowed to recover for the indicated time. Cells were lysed and analyzed by alkaline Comet assay. Comet tail moment averages are shown. One of three replicate experiments is shown. Error bars represent s.d. h Endogenous GFI1 protein was immunoprecipitated in SupT1 cells. Co-precipitated proteins were separated by SDS–PAGE and blotted for MRE11 and PRMT1. i Endogenous GFI1 protein was immunoprecipitated in Jurkat cells. Co-precipitated proteins were separated by SDS–PAGE and blotted for MRE11 and PRMT1 Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/s41467-018-03817-5), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human GFI-1 by Western Blot

GFI1 activities are independent of DNA damage. a GFI1-Flag fusion protein was immunoprecipitated in 293T cells treated with 5 Gy IR and allowed to recover for the indicated amount of time. Extracts were separated by SDS–PAGE and blotted for the indicated proteins. b SupT1 cells were spread on glass slides 15 min and 1 h after irradiation using a Cytospin, stained for endogenous Gfi1 and gamma-H2AX and visualized for immunofluorescence by confocal microscopy. Control cells stained without primary antibody but with secondary antibodies are shown. c U2OS cells carrying a LacO array and expressing a LacR-Fok1-mCherry endonuclease were transfected with a vector expressing the GFI1-GFP fusion protein. These cells were plated on cover glass, stained for gamma-H2AX and visualized for immunofluorescence by confocal microscopy. d U2OS cells expressing a GFI1-GFP fusion protein were exposed to 405 nm UV micro-irradiation and the recruitment of the GFI1-GFP fusion protein to the site of damage was quantified by confocal microscopy. Average signal intensity is shown with error bars representing s.d. Recruitment of Ku80-mRuby2 fusion protein and GFP protein are shown as controls. Representative images of selected time points are shown on the right. Scale bar represents 10 μm Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/s41467-018-03817-5), licensed under a CC-BY license. Not internally tested by R&D Systems.