(A) Organellar maps were prepared from untreatedHeLa cells (control, left side), and from cells following continuous stimulation with EGF for 20 min (+EGF, right side). The individual maps from triplicate biological repeats are shown, visualized by PCA. Organellar clusters are colour coded as in Figure 2. Major translocating proteins are shown as unique symbols. CBL and UBASH3B were identified in only one of the +EGF maps; they are mostly cytosolic before EGF treatment, and hence not identified in control maps. (B) Detection of EGF-induced global profile changes. Nuclear, membrane and cytosolic fractions from the experiments described in A) were subjected to mass-spectrometric analysis using label-free quantification (LFQ). Mean Log2 LFQ values from triplicate control experiments were subtracted from triplicate EGF stimulation experiments and plotted against Student’s (two-sided) t-test p-value for that difference (a ‘volcano’ plot). Proteins that increase in abundance in the relevant compartment following EGF stimulation are found on the right-hand side of the plots. Proteins undergoing significant translocations are shown in red, based on cut-offs determined as follows. First, the protein must show a minimum two-fold change in abundance (absolute log-difference >1). Second, the protein must constitute at least 10% of the total pool, either before or after EGF stimulation, in the compartment where it is shown to be changing (as determined from the protein’s global intensity profile; see Figure 1A). Finally, the p-value cut-off was FDR-controlled using the six control maps generated in Figure 2—figure supplement 2A as a mock experiment, in which no true positives would be expected. Three maps were assigned as mock-treated, three as control. For each compartment, a p-value cut-off was chosen such that no false positives would be detected in the mock experiment, but changes could still be detected in the genuine experiment (FDR = 0). This was possible for cytosolic and membrane fractions (-log10 p=2.0 and 3.1, respectively). In the case of the nucleus (-log10 p=2.6), two false positives are expected among the 13 positives (FDR ≈ 15%). Two relevant changes (shown in grey) narrowly missed significance with our extremely stringent cut-offs (SHC1 in the organellar fraction, and MAPK1 in the nuclear fraction). While their p-values were sufficiently high to reach significance, their fold-changes were just below two.