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. 2010 Jan 20;30(3):1166–1175. doi: 10.1523/JNEUROSCI.3944-09.2010

Figure 5.

Figure 5.

Rapamycin prevents RTP801 protein upregulation by MPTP in vivo and protects substantia nigral neurons from MPTP in living mice. a, Mice were injected with vehicle, rapamycin, MPTP, or rapamycin plus MPTP, as described in Materials and Methods for the acute regimen, and killed after 24 h. Ventral midbrains were dissected and homogenized in lysis buffer, and the homogenates were subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with RTP801 antibody and reprobed with an antibody for total ERK1/2 as a loading control. Film resulting from the immunoblot was scanned, and relative densities of RTP801 bands were normalized in each case to the densities of the corresponding ERK1/2 signals. Values (in arbitrary units) for RTP801 are expressed as mean ± SEM for at least three mice per condition. *p < 0.01 versus mice injected with vehicle; ψ p < 0.01 versus mice injected with MPTP. M.W., Molecular weight. b, Mice subjected to the acute regimen as indicated were killed 7 d after the last MPTP injection. Midbrains were cryosectioned, immunostained with tyrosine hydroxylase antibody, and subjected to Nissl staining. Unbiased stereology was used to determine the total numbers of TH-positive neurons in the substantia nigra. Results represent mean ± SEM of total number of TH-positive cells per substantia nigra (right and left) for at least seven mice per each condition. **p < 0.001 versus mice injected with vehicle; ++ p < 0.01 versus mice injected with rapamycin plus MPTP. Bottom shows representative micrographs of SN stained with TH antibody in comparable sections for each condition. c, Mice subjected to the subacute MPTP regimen as indicated were killed 2 d after the last injection. Midbrains were cryosectioned, immunostained lightly with tyrosine hydroxylase antibody, and subjected to Nissl staining to visualize condensed nuclei (with fragmented chromatin). Results represent mean ± SEM of total number of apoptotic nuclei per midbrain for at least four mice per each condition. ***p < 0.001 versus mice injected with MPTP alone.