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. 2014 Jan 21;6(3):398–413. doi: 10.1002/emmm.201302649

Figure 3.

Figure 3

Absence of ASM and SM modulation alter the levels and activity of RhoA and its effectors in synaptosomes.
  1. Western blot of RhoA and tubulin levels in total (a) and membrane extracts (b) from wt and ASMko synaptosomes. Graphs show mean ± s.d. of RhoA levels in ASMko conditions normalized to tubulin and referred to wt levels that were considered as 1 (n = 3, *Ptotal RhoA = 0.04, *Pmembrane RhoA = 0.008).
  2. (a) Activity of RhoA in wt and ASMko synaptosomes determined by the Rhotekin binding assay. Tubulin is shown as loading control. Graph shows mean ± s.d. of the ratio of Rhotekin-bound (active) RhoA to total RhoA (n = 3, *P = 0.025). (b) Western blots of ROCK, ProfilinIIa and tubulin levels in membrane extracts from wt and ASMko synaptosomes. Graphs show mean ± s.d. of ROCK (*P = 0.017) or ProfilinIIa (*P = 0.033) levels in ASMko conditions normalized to tubulin and referred to wt levels that were considered as 1 (n = 3).
  3. (a) SM levels (nmol/mg protein) in wt synaptosomes treated or not with SM. Graph shows mean ± s.d. in treated synaptosomes referred to non treated that were considered as 1 (n = 3, **P = 0.019). (b, c, d) Western blots of RhoA (b), ROCK (c) and ProfilinIIa (d) levels in supernatants (S) and pellets (P) after 100,000 g centrifugation of wt synaptosomes treated or not with SM. Graphs show mean ± s.d. of each protein ratio pellet/supernatant in treated samples referred to non-treated that were considered as 1 (n = 3; *PRhoASM = 0.029, ***PROCKSM = 0.0009, **PprofilinIIaSM= 0.008).