Journal: The Journal of Biological Chemistry
Article Title: Functional and biochemical characterization of a T cell-associated anti-apoptotic protein, GIMAP6
Figure Lengend Snippet: GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor 488 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
Article Snippet: Alexa Fluor 647 or 488 goat anti-rabbit IgG (Life Technologies) and Alexa Fluor 488 or 594 goat anti-mouse IgG (Life Technologies) was used.
Techniques: Expressing, Derivative Assay, RNA Expression, Real-time Polymerase Chain Reaction, Standard Deviation, Confocal Laser Scanning Microscopy, Marker, Immunofluorescence, Microscopy