Stalling at <i>RFB</i> in the absence of Top2 and Sgs1 leads to checkpoint activation whereas stalling at other rDNA barriers causes X-spike formation.

<p>(A) Genomic DNA was isolated from <i>sgs1Δtop2</i>^<i>ts</i><i>pif1Δ</i> at the indicated time points after release from α-factor and processed for 2D gel analysis and Southern blotting using either probe P1, recognizing <i>Bgl</i>IIB (upper panel), or probe P2, recognizing <i>Bgl</i>IIA (lower panel). Quantification of the Xs to Ys obtained with the <i>Bgl</i>IIB fragment is shown to the right, where the Xs/Ys at time point 0 is set to 1. Error bars represent STDEV from three independent experiments. FACS profiles are shown to the right. (B) ISA analysis of Rad53 was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005697#pgen.1005697.g001" target="_blank">Fig 1</a> with protein isolated from <i>sgs1Δtop2</i>^<i>ts</i> and <i>sgs1Δtop2</i>^<i>ts</i><i>pif1Δ</i> cells.