Figure 5.
Differential interaction of signaling proteins with dynein observed in SOD1G93A but not in Loa mice compared with controls. A–C, Sciatic nerve extracts from 85-d-old SOD1G93A (S), 12-month Loa (L), and age-matched n.Tg and wild-type (W) mice were used in DIC pull-down assays followed by Western blot analysis for different signaling proteins. Positive controls (Post. CTRL) for the pull-down assay were the p150Glued and p50 (dynamitin) subunits of dynactin; GAPDH was used as a negative control (Neg. CTRL). D–F, P-Trk, P-Erk1/2, P-Erk5, and Bim all showed a decreased association with dynein in SOD1G93A but not in Loa mice. G–I, Increased association with dynein was observed for activated caspase-8, P-JNK, and p75 cleavage fragment in SOD1G93A mice, but no significant changes in these dynein cargos were observed in Loa mice. PD, DIC pull-down; CTRL, control beads; CLV, cleavage; FL, full length; CTF, C-terminal fragment; ICD, intracellular domain; Casp8, caspase-8. J, K, Differential association of vesicular signaling proteins observed in SOD1G93A but not in Loa mice compared with wild-type controls (J). Vesicle extracts from mSOD1, Loa, and wild-type mice were subjected to Western blot analysis for differences between pTrk and p75. Synaptotagmin was used as loading control. Whereas there was a decrease in P-Trk levels (>75%; p < 0.01) and an increase in p75 cleavage fragments in the mSOD1 mice vesicles compared with control, the levels of P-Trk and p75 were the same in Loa mice compared with WT control. Blots are from representative experiments (J) and the graphs are averages of relative changes (1 = no relative change) of dynein association with specific cargo from ≥3 independent experiments ± SEM, compared with control mice (K). S, Soup; P, pellet; 0.6, 0.6 m sucrose; V, vesicle extracts; 1.5, 1.5 m sucrose; Syn, synaptotagmin.