Skip to main content
. 2007 Aug 8;104(33):13361–13366. doi: 10.1073/pnas.0701243104

Fig. 4.

Fig. 4.

DRG neurons death upon NT-3 loss depends on the proapoptotic activity of TrkC. (A and B) TrkC IC D641N acts as a dominant-negative mutant of TrkC. HEK293T cells were transfected with the mock plasmid (Mock), the TrkC expression plasmid together with the mock plasmid, or the TrkC IC D641N expression plasmid. The dominant-negative effect of TrkC IC D641N was measured by trypan blue exclusion (A) or a caspase activity assay, as in Fig. 1B (B). Standard deviations are indicated (n = 3). (C) 13.S.24 neuroblasts were transfected with TrkC wild type or TrkC kinase-dead (TrkCD679N), or cotransfected with TrkC and the dominant-negative mutant TrkC IC D641N, in the presence or absence of 10 ng/ml NT-3 and with or without the addition of a PI3K inhibitor (LY294002, 10 μM) or a MEK inhibitor (U0126, 10 μM). Akt and Erk phosphorylation was visualized by Western blot with an anti-phosphoAkt and an anti-phosphoErk antibody, respectively. The levels of Akt and Erk kinases were shown by reprobing the membrane with an anti-total Akt antibody or an anti-total Erk antibody, respectively. TrkC immunoblot is also shown. Similar results were obtained by using the FACE-Akt ELISA (Active Motif, Carlsbad, CA). (D and E) Sensory neurons were maintained with NT-3 or NGF, microinjected with a mock plasmid (Mock) or the plasmids encoding TrkC IC D641N (D) or kinase dead TrkC IC D641N/D679N, the dominant-negative mutant of Ret (i.e., Ret IC D707N) or TrkC IC (E), and grown further without NT-3 or NGF. Living neurons were counted 72 h later and expressed as the percentage of initially injected neurons. Experiments shown in D and E were performed separately. (F) Same as D and E: sensory neurons were maintained with NT-3, microinjected with endogenous TrkC siRNA and either TrkC or TrkC D495N/D641N, and grown further in the absence of NT-3. Living neurons were counted 72 h later and expressed as the percentage of initially injected neurons. The standard errors of the means are shown (n = 3). (G) Same as in C, except that Akt/Erk phosphorylation was measured in 13.S.24 cells transfected with TrkC or TrkC D495N/D641N.