DR1_a generated after photocleavage of Y1MxN its stable at 4°C.

<p>(A) 150 nM DR1-Y1MxN was exposed to UV light on ice during 20 minutes and incubated on ice for different periods of time: not incubated (blue), 15 minutes (green), 30 minutes (red) and 60 minutes (pink). As a control, DR1-Y1MxN not exposed to UV light and kept always on ice was also tested (black trace). Then it was mixed with 25 nM labeled HA and binding to DR1 was measured by fluorescence polarization. The concentration of labeled HA bound to DR1 is plotted vs. time. Each data point shown in the plot is the mean of duplicate samples from one of two independent experiments. (B) 150 nM DR1 empty (left panel) or DR1-Y1MxN (right panel) were not exposed (black trace) or exposed to UV light for 5 (red and orange traces) or 20 minutes (green and blue traces) on ice. 25 nM labeled HA was either present during the UV exposure (“+HA” samples, orange and blue traces), or added after the exposure (“-HA” samples, red and green traces) and binding of labeled HA to DR1 was measured by fluorescence polarization. The concentration of labeled HA bound to DR1 is plotted versus time. Each data point shown in the plot is the mean of triplicate samples from one of three independent experiments. (C) The initial peptide binding rates were calculated as the slope of a linear fit to the initial time points of the peptide binding data shown in panel B and plotted as a bar graph. The bars represent the mean and the error bars the standard deviation of the initial rates calculated from three independent experiments done with triplicate samples.