Fig. 4.

Increased RhoA-myosin II pathway in Mtmr2 KO nerves. (A) Pull-down assay from Mtmr2 KO and control sciatic nerves at P2 and P60 using GST-RBD/Rhotekin as a bait to detect active GTP-bound RhoA. GTPγS and GDP were used as control to check the GST-RBD/Rhotekin recombinant protein. Representative data from two independent experiments at P2 and five at P60 (SI Appendix, Fig. S4 A and B). (A′) Quantification of the active RhoA-GTP GST-bound fraction at P60 in control and mutant nerves, n = 5 independent experiments, one-sample t test, *P = 0.0438, t = 2.907, df = 4. (B) Western blot analysis shows an apparent increase of p-MLCII levels in each sciatic nerve pool of Mtmr2 KO mice analyzed as compared to controls. Three independent experiments. (C) Western blot analysis indicates that phosphorylation of the MYPT1 phosphatase downstream of RhoA is increased in Mtmr2 KO sciatic nerve lysates at P20, P30, and P60, and quantified in C′, n = 7 animals per genotype in four independent experiments, one-sample t test, *P = 0.0264, t = 2.926, df = 6 (SI Appendix, Fig. S4C). (D) Western blot analysis indicates that phosphorylation of the MYPT1 phosphatase is increased in Mtmr2^{Floxed/Floxed}; P0-Cre (the conditional KO of Mtmr2 in Schwann cells, cKO^SC) sciatic nerve lysates at P60, and quantified in D′, n = 3 animals per genotype in two independent experiments, one-sample t test, one tail, *P = 0.0486, t = 2.967, df = 2 (SI Appendix, Fig. S4D). (E) Phosphorylation levels of MYPT1 are similar between control and Pmp22^+/− mutant nerves, n = 3 animals per genotype in two independent experiments, one-sample t test, P = 0.2478, t = 1.615, df = 2 (SI Appendix, Fig. S4E).