Fig. 5.

SHP-2 interacts with the SH-2 binding motif of MyD88.

A) IL-1β stimulation (10 ng/ml, 2 min) of EC induced MyD88 association with SHP-2 (*p < 0.05, student's t-test, n = 6 each 4–5 fields of view), as measured by a proximity ligation assay (PLA). B) HA-tagged MyD88 WT interacted with endogenous SHP-2 upon IL-1β stimulation, whereas HA-tagged MyD88 YF (mutation of Y257 in the SH-2 binding motif) did not associate with SHP-2 (*p < 0.05, 1-way ANOVA, n = 7 each 4 fields of view). C) PLA with myc-tagged SHP-2 WT, CS and EA revealed interaction between MyD88 and SHP-2 WT upon IL-1β. This interaction was increased between SHP-2 CS and MyD88, but impaired between SHP-2 EA and MyD88 (p < 0.05, 1-way ANOVA, n = 6 each 4–5 fields of view). D) IL-1β stimulation induced interaction between MyD88 and the p85 subunit of the PI3-K (*p < 0.05, student's t-test, n = 6 each 4 fields of view). E) IL-1β-mediated MyD88/p85 interaction was enhanced in EC expressing the substrate trapping and inactive mutant SHP-2 CS compared to SHP-2 WT. In contrast, interaction of MyD88 and p85 was prevented in cells expressing SHP-2 EA (*p > 0.05, 1-way ANOVA, n = 6 each 4 fields of view).

Representative images of all PLA experiments are shown to the right of the respective graph. Specific interaction spots are shown in the left panel. Merge images (right): PLA spots (red), nuclear staining with DAPI (blue) and actin staining with phalloidin AF488 (green). Scale bar: 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)