Figure 5

Secretome analysis of RAP-treated HEK293 cells. (A) Topology of all the proteins detected in the secretome of RAP-treated HEK293 cells. (B) Volcano plot showing the −log10 of p-values versus the log2 of protein ratio between HEK293 cells in the presence and absence of RAP (left). RAP itself was also identified and is shown in the plot under the gene name ‘LRPAP1’. From this analysis, a volcano plot showing only secreted proteins was extracted (right). The horizontal solid line indicates the –log10(p-value) of 1.3 (corresponding to a p-value of 0.05). Proteins above this line are considered significantly regulated (red dots for upregulated proteins, light blue dots for downregulated proteins, (n = 6). (C) Venn diagram showing secreted proteins that are upregulated in either TIMP-3/HEK or RAP-treated HEK293 secretomes (p-values < 0.05, and proteins found only in TIMP-3/HEK or RAP-treated secretomes, but not in the control). In the intersection between the two diagrams are listed proteins upregulated in both secretomes. (D–G) Immunoblots (D,F) and their respective quantifications (E–G) showing that addition of 500 nM RAP to HEK293 cells does not alter shedding of APP (D,E) and EphA4 (F,G, displayed bands were run on the same blot but not directly next to each other) (densitometric quantifications shown as mean values ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.005, Student’s t-test; 2 independent experiments are displayed out of 6 quantified, n = 6).