|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. Morphometric analysis of RalA-depleted cells. In neurons with a polarised morphology, RalA knockdown causes a modest decrease in total neurite length (A, mean±s.e.m.: control siRNA, 348.45±8.20; RalA siRNA, 265.32±8.58; ***P<0.001), does not affect minor neurite branch density (B, mean±s.e.m.: control siRNA, 1.40±0.11; RalA siRNA, 1.26±0.18) but decreases axon branching (C, mean±s.e.m.: control siRNA, 1.68±0.07; RalA siRNA, 1.09±0.09; ***P<0.001). In unpolarised cells, lack of RalA impairs branching (D) (mean±s.e.m.: control siRNA, 1.60±0.06; RalA siRNA, 1.00±0.05; ***P<0.001). (E) Fluorescence ratiometric analysis to evaluate protein accumulation at axon tips. (Left) Example of stage 3 neurons incubated with the cytoplasmic tracer CMFDA and stained for p38 or Sec6. Sec6 accumulates at the tip of the presumptive axon, while the negative control protein p38 does not. Insets show a magnification of the axon growth cone. Bar, 20 µm. (Right) Average ratio of the fluorescence/CMFDA value in axon tips to the other cell neurites for each of the indicated proteins. Proteins were considered accumulated at the axon tip if the fluorescence/CMFDA ratiometric value at the axon tip was 1.5 fold higher that that at the tip of the other neurites (mean±s.e.m.: p38, 1.15±0.03; RalA, 1.61±0.08; Sec6, 2.32±0.11; Sec8, 1.27±0.05; Exo84, 1.75±0.07; ***P<0.001).
Fig. S2. Morphometric analysis of polarised neurons lacking exocyst subunits. Total neurite length (A, mean±s.e.m.: control siRNA, 348.45±8.20; Sec6 siRNA, 274.46±10.28; Sec8 siRNA, 240.70±9.60; Exo84 siRNA, 208.92±25.35; ***P<0.001), and both minor neurite (B, mean±s.e.m.: control siRNA, 1.40±0.11; Sec6 siRNA, 0.88±0.14; Sec8 siRNA, 0.53±0.12; Exo84 siRNA, 0.53±0.17; ***P<0.001) and axon branch density (C) are significantly decreased (mean±s.e.m.: control siRNA, 1.68±0.07; Sec6 siRNA, 0.87±0.10; Sec8 siRNA, 0.86±0.09; Exo84 siRNA, 0.31±0.08; ***P<0.001).
Fig. S3. Western blots of extracts from neurons nucleofected with the indicated siRNA and lysed 48 hours after plating.
| ||||||||||||||||||||
