L* interferes with 2-5A binding to mouse RNase L.

<p>A. Lysates of 293T cells overexpressing Flag-RNase L and L* were treated with increasing concentrations of 2-5A (0, 2, 4 and 8 μM ATP equivalent of 2-5A) before incubation, RNA extraction and analysis on agarose gel. Arrowheads indicate RNase L-generated rRNA degradation products upon treatment with high concentration of 2-5A, arising despite the presence of L*. B-C. Biotin-labeled 2-5A was immobilized on streptavidin-coated SPR gold chips. (B) Mouse (mu)RNase L (2.5 μM) or (C) human (hu)RNase L (2.5 μM) were injected in the absence or presence of different concentrations of L* as indicated. D. His<sub>6</sub>-L* binding was immobilized on Ni-NTA chips and mouse RNase L (2.5 μM) was injected in the absence or presence of different concentrations of 2-5A as indicated. RU, response units. E. Kinetic parameters of interactions between mouse RNase L and L* or 2-5A. F-G. Histograms showing mean relative fluorescence units (RFU) resulting from degradation of a FRET RNA probe by RNase L. Experiments were repeated twice in triplicates. F. Mouse RNase L (100 nM) was pre-incubated with mock (circle) or 10μM (triangle) of L* followed by addition of varying concentration of 2-5A p3A3. Graph showing the mean and SEM of RNase L activity and table showing inferred Vmax (maximum velocity), K50 (concentration for half activation) and statistical significance of the differences between the two conditions (unpaired t-test). Ns: non-significant G. Mouse RNase L was either pre-incubated with varying concentration of L* (circle) followed by addition of 3nM of 2-5A or pre-incubated with 3 nM 2-5A followed by addition of varying concentration of L* (triangle). Graph showing mean and SEM of RNase L activity and table showing inferred IC<sub>50</sub> (half maximal inhibitory concentration) and statistical significance of the difference between the two conditions (unpaired t-test).</p>