Figure 4.

PLD enzymes create TLR7 second binding pocket fragments

(A) CAL-1 cells of indicated genotypes were unstimulated or stimulated with pR, RNA40^S, RNA40^O, RNA9.2s^O, R848, 2′,3′-cGMP (0.5 mM), CpG^O, and CpG^S. After 16 h, IFN-β release was determined by enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± SEM of n = 3 independent experiments. Statistical analysis was conducted by two-way ANOVA with Dunnett’s multiple comparison tests. Note that the WT data are identical with the ones shown in Figure 1A.

(B) Urea gels of RNA9.2s^O digested with PLD4 (+ = 25 nM, ++ = 250 nM) over time. One representative gel of three independent experiments is shown.

(C) Urea gels of RNA9.2s^O digested with PLD3 (+ = 0.39 nM, ++ = 1.56 nM) over time. One representative gel of two independent experiments is shown.

(D) Urea gel of RNA9.2s^O (1 μg) incubated with PLD4 (++ = 250 nM, + = 25 nM) for 2 h. One out of three independent experiments is shown.

(E) LC-HRMS total ion current (TIC) chromatogram of RNA9.2s^O-derived ORN fragments after digestion with PLD4 (250 nM) for 2 h.

(F) Calculated and found masses (m/z) of RNA9.2s-derived ORN fragments after digestion with PLD4 (250 nM) for 2 h.

(G) Urea gel of indicated substrates digested with PLD4 (++ = 250 nM, + =25 nM). One out of two independent experiments is shown.

(H) LC-MS/MS analysis of depicted substrates digested with PLD4 (250 nM) for 20 min. Data were normalized to the amount of the different nucleosides present in the sequence and are depicted as mean ± SEM of n = 3 independent experiments.

(I) CAL-1 cells of indicated genotypes were unstimulated or stimulated with 2′,3′-cGMP (0.5 mM), short ORNs, or ORNs in combination with 2′,3′-cGMP (0.5 mM). Data are depicted as mean + SEM of n = 3 independent experiments. Statistical analysis was conducted by two-way ANOVA (left panel) or one-way ANOVA (right panel) with Dunnett’s multiple comparison tests.