Fig 2: Notch is a target of tankyrase. a Schematic diagram of Notch1/2. Shaded box; RAM (RPBJ association molecule) domain that binds RPBJ (recombining binding protein suppressor of hairless). PM plasma membrane. Alignment of tankyrase-binding sites; identical amino acids in black. The acidic residue at the 8th position of the tankyrase-binding motif indicates an 8-amino-acid motif. b Immunoblot analysis showing Notch2 is stabilized in RNF146 siRNA-treated HEK293T cells. c Endogenous tankyrase and Flag-Notch2 coimmunoprecipitate. Immunoblot analysis of HEK293T cells transfected with Flag-Notch2 and immunoprecipitated with tankyrase IgG. d Endogenous tankyrase and Flag-Notch2 coimmunoprecipitate, dependent on the Notch2 tankyrase-binding site. Immunoblot analysis of HEK293T cells transfected with vector, Flag-Notch2 WT, or Flag-Notch2 Mut and immunoprecipitated with anti-tankyrase IgG. e Notch2 is a target for ADP-ribosylation by tankyrase 1. Flag-Notch2 or Myc-TRF1 immunoprecipitates from HEK293T cells were incubated with 32P-NAD+ and recombinant tankyrase 1, fractionated by SDS-PAGE and visualized by Coomassie blue (left panel; asterisks indicate FlagNotch2 and MycTRF1) and autoradiography (right panel). f Notch2 is increased at the plasma membrane in TNKS DKO cells. Immunofluorescence analysis of formaldehyde-fixed HEK293T WT, TNKS DKO, or TNKS DKO expressing tankyrase 1 and 2 (T1T2) cells stained with anti-Notch2 antibodies (green). DNA was stained with DAPI (blue). Scale bar, 5 µm. g Notch2 is increased at the plasma membrane in HeLa cells treated with inhibitors to tankyrase (Ti8) or ?-secretase (DAPT). Immunofluorescence analysis was performed as described in f. h, i Expression of the Notch target gene Nestin is reduced in h TNKS DKO cells and in i inhibitor-treated HEK293T cells. mRNA levels were quantified using qRT-PCR. Average of two to four independent experiments (with three technical replicates each) ± SD. *p = 0.05, **p = 0.01, Student's unpaired t-test. j–l Notch2 stability is regulated by TNKS1, not TNKS2. j Immunofluorescence analysis of TNKS1 and TNKS2 DKO cells was performed as in f. k Immunoblot analysis of HEK293T WT and TNKS KO cells lines. l graphical representation of the abundance in TNKS KO cells relative to WT. Average of two independent experiments ± SEM. *p = 0.05, Student's unpaired t-test

Fig 3: 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).