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. 2017 Mar 22;8:320. doi: 10.3389/fimmu.2017.00320

Figure 6.

Figure 6

Rik inhibits nuclear translocation of Notch2 intracellular domain (NICD2). (A) mRNA levels of HES1, HES6, and HEY1 in CD8+ T cells. Lentivirus-transduced CD8+ T cells were stimulated in vitro with immobilized delta-like 1 (DLL1) for 4 h before quantitative polymerase chain reaction. L-S: CD8+ T cells transduced with scramble lentiviruses. L-R: CD8+ T cells transduced with Rik-expressing lentiviruses. V: vehicle. D: DLL1. N = 3 per group. (B) Expression of Notch1, Notch2, NICD1, and NICD2 in the whole cell lysates after 1-h stimulation with DLL1. This is a representative of two independent experiments. (C,D) Cell surface expression of Notch1 and Notch2 after 4-h stimulation with DLL1. Representative histograms are shown in panel (C), and statistics of mean fluorescent intensity is shown in panel (D). N = 3 per group. (E,F) Expression of NICD1 and NICD2 in the cytosol and nucleus of CD8+ T cells after 1-h stimulation with DLL1. Representative Immunoblot images are shown in panel (E), and statistical analysis is shown in panel (F). N = 4 per group. (G) Fluorescent microscopy of Notch2. The polyclonal anti-Notch2 antibody recognized both intact Notch2 and NICD2. N: naïve CD8+ T cells. D: DLL1-stimulated CD8+ T cells. (H) Immunoprecipitation of Rik and detection of NICD2. This is a representative of two independent experiments. (I) Nuclear NICD2 levels in isolated intratumoral CD8+ T cells from 3 individual mouse of each group. Left panel: representative Immunoblot image. Right panel: statistics of nuclear NICD2 levels. L-S: mice receiving control CD8+ T cells. L-R: mice receiving Rik-overexpressing CD8+ T cells (**p < 0.01; ***p < 0.001).