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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Cancer Immunol Res. 2014 May 15;2(8):800–811. doi: 10.1158/2326-6066.CIR-14-0021

Figure 4. Transgenic N1IC regulates granzyme B expression by amplification of canonical and non-canonical pathways.

Figure 4

(A) ChIP assays to monitor endogenous binding of RBP-J and NF-κB to granzyme B promoter were assessed in N1IC and N1ICf/f CD8+ T cells cultured with or without siinfekl for 48 hours. Results represent mean ± SD from 3 independent experiments. ***, P < 0.001. (B) Immunoprecipitations were achieved using 200 μg of protein extracts from activated N1IC and N1ICf/f CD8+ T cells and 2 μg anti-Notch-1 or control IgG antibodies. After overnight incubation, protein G plus-captured complexes were analyzed for Notch-1 (cleaved and transgenic), RBP-J, and NF-κB p65 by western blot. As input controls, we used 10 μg of extracts from each experimental group before immunoprecipitation. Representative illustrations are from 2 experiments. (C) Kinetics for RBP-J and NF-κB p65 in activated N1IC or N1ICf/f cells. Representative blotting from 3 independent repeats. (D) N1IC and N1ICf/f cells were activated with siinfekl in the presence or absence of PTDC (150 nM). Granzyme B levels were tested 72 hours later. Representative illustrations from 3 independent experiments are shown.