Visualization of UmuC redistribution by cross-correlation analysis and Western blotting.

<p>(A) Cross-correlation analysis of LacY-eYFP and UmuC-mKate2 signals from time-lapse measurements (EAW191 containing pBAD-LacY-eYFP). Distance-dependent correlation values between LacY-eYFP and UmuC-mKate2 signals are presented as 2D contour plots. Blue areas indicate low correlation, whereas red areas indicate high correlation. Image pairs containing membrane-associated UmuC-mKate2 foci, thus co-localizing with membrane-defining LacY-eYFP signals, produce sharp cross-correlation peaks at lag = 0 and ± 0.8 μm. Image pairs containing cytosolic UmuC-mKate2 foci produce broader cross-correlation peaks at values between 0 and 0.8 μm. Red dotted lines mark the position of the secondary peaks in time and highlight a time-dependent trend towards lag values < 0.8 μm. A peak-enhancing filter was applied to fluorescence images prior to autocorrelation analysis. (B) UmuD, UmuD' and UmuC expressed from the <i>recA</i>⁺<i>lexA</i>⁺ strain, RW118, after exposure to 30 Jm^-2 was monitored in whole cell extracts (WCE) and soluble fraction (SOL) at various time points after irradiation. The protein that is slightly smaller than UmuC and cross-reacts with the UmuC antibodies is indicated with an asterisk to the right of each lane. When detectible, UmuC is indicated by an arrow on the left of each lane. The UmuD and UmuD' proteins are indicated on the right hand side of the image