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. 2017 Oct 17;47(4):739–751.e5. doi: 10.1016/j.immuni.2017.09.015

Figure 7.

Figure 7

HpARI Binds Nuclear DNA, Tethering IL-33 within Necrotic Cells

(A) Live (top panels) or freeze-thawed (bottom panels) CMT-64 cells were incubated for 1 hr at 37°C with 5 μg/ml HpARI_mCherry.

(B) HpARI_mCherry-stained freeze-thawed CMT-64 cells, with Hoechst 33342 nuclear co-stain.

(C) Freeze-thawed CMT-64 or HEK293T cells were stained with HpARI_mCherry with 100 U/ml DNAse I.

(D) Murine IL-33 western blot densitometry of BAL taken 15 min after Alternaria allergen, HpARI and DNAse (100 U) intranasal administration.

(E) Murine IL-33 levels (ELISA) IL-33 in BAL fluid from mice described in (D)

(F) Gel shift assay of linearised plasmid DNA, incubated with 100, 50 or 25 pmol of HpARI, CCP1/2 or CCP2/3 truncated proteins.

(G) Murine IL-33 western blot densitometry of BAL taken 15 min after Alternaria allergen, HpARI or CCP1/2 or CCP2/3 HpARI truncated proteins intranasal administration.

(H) Murine IL-33 levels (ELISA) in BAL from mice described in (G).

All data representative of at least 2 repeat experiments. Data in (D) and (E) shows mean and SEM of 3 pooled experiments, data log-transformed for statistical analysis to equalize variances. Scale bars = 100 μm. Error bars show SEM.