RAB27A Antibody - #DF6702

Fig. 4 CD45 overexpressing tumor cells were resistant to T cell killing. a T cell killing effect was tested in DLD1 cells that had been indirectly pre-cocultured with Jurkat cells. For the evaluation of T cell killing, PBMCs (effector; E) were incubated with CD45− or CD45+ DLD1 cells (tumor cell; T) at a E:T ratio of 2:1. b T cell cytotoxicity assay of DLD1 cells (preincubated with PBS or Jurkat cell-derived EVs) in co-culture with PBMCs after the addition of OKT3 (1 μg/mL) at various E:T ratios. The images on the left are representative images of survived DLD1 cells stained with crystal violet, and the graph on the right is a line graph showing the relative intensity of survived DLD1 cells. c, d Immunoblot analysis showing CD45 expression after CRISPR Cas9 knockout of PTPRC or RAB27A knockdown in Jurkat cells. e Bar graph showing EVs-derived CD45 levels from supernatant of Jurkat cells by ELISA assay. f T cell cytotoxicity assay of DLD1 cells (preincubated with PBS or Jurkat cell-derived EVs (EVs from Jurkat-Ctrl, Jurkat-sgCD45 or Jurkat-shRAB27A cells) in co-culture with PBMCs after the addition of OKT3 (1 μg/mL) at different E:T ratios. g Schematic diagram showing the different CD45 isoforms tested in the study. h Immunoblot analysis of DLD1 cells overexpressing different CD45 isoforms. i Immunofluorescence imaging of membrane CD45-GFP on DLD1 cells. Scale bar = 5 μm. j Comparison of CD45 expression on DLD1 cells and Jurkat cells. k Proliferation assay of DLD1 cells overexpressing different isoforms of CD45. l, m T cell cytotoxicity assay of DLD1 cells with or without CD45 overexpression co-cultured with PBMCs in the presence of pre-coated OKT3 (1 μg/mL) at different E:T ratios. n–p Quantitative analysis of IFN-γ, TNF-α, and IL-2 secretion in the co-culture system of T cell cytotoxicity assay with DLD1 cells. q Proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8+ T cells in the co-culture system of T cell cytotoxicity assay with DLD1 cells. r Immunoblot analysis of Caco2 cells overexpressing different CD45 isoforms. s, t T cell cytotoxicity assay of Caco2 cells with or without CD45 overexpression co-cultured with PBMCs in the presence of pre-coated OKT3 (1 μg/mL) at different ratios. u–w Quantitative analysis of IFN-γ, TNF-α, and IL-2 secretion in the co-culture system of T cell cytotoxicity assay with Caco2 cells. x Proliferation of CFSE-labeled CD8+ T cells in the co-culture system of T cell cytotoxicity assay with Caco2 cells. y NK cell sorting strategy from WBCs. z Lysis percentage of NK cell killing Caco2 cells with density diluted NK cells at NK: Tumor ratios of 80: 8 to 5: 8. All bar graph data are presented as means ± SEM. *P