Phosphoproteomics of a Single, Injured Sciatic Nerve in Adult Mice Reveals that pS96 Is Specifically Detected at Regenerating Axons
(A) Schematic of phosphoproteomics procedure for a single injured sciatic nerve. The crushed region of a sciatic nerve was excised and divided into 1 cm segments, which were prepared for SDS-PAGE. The band corresponding to GAP-43 was cut out, trypsinized in-gel, and subjected to MS analysis. *, molecular mass marker.
(B) Shotgun phosphoproteomics analysis of a single crushed nerve. Note that pS96 was detected in three of five injured samples, and another JNK-dependent site, pS142 (Figure 3C), was also detected twice. By contrast, no phosphorylated peptides of GAP-43 were detected in intact nerves.
(C) MS quantification of pS96 using HR-MRM to compare crushed (Crush) and intact sciatic nerves on day 3 after injury. Crush represents the regenerating axons. **p < 0.01 (Student's t test). All data are shown as means ± SD.