Figure 5.

Hsp90 inhibition induces protein expression and signaling changes in mouse brain. Mice were injected i.c.v. with vehicle or 0.5 nmol of 17-AAG, allowed to recover for 24 h, and then injected i.c.v. with vehicle (Veh) or 0.1 nmol of DAMGO for 10 min. Brains were extracted and analyzed by Western blot analysis as reported under “Experimental procedures.” All data are reported as the mean ± S.D. A, representative blots from each protein target are shown, with the treatment and molecular weight markers indicated as described in the legend for Fig. 1. PAG (B) and caudal brain stem (pons and medulla) (C) protein expression changes are indicated. The signal for each target is normalized to GAPDH and further normalized to the vehicle control for each. MOR and βarr2 in the PAG were measured by immunoprecipitation (see “Experimental procedures”). *, p < 0.05 versus same target vehicle control by unpaired 2-tailed t test. The reported sample size of the individual mice consists of two technical replicates performed by two different experimenters at different institutions. Only Hsp70 expression was altered by 17-AAG treatment in both brain regions, contrary to some of the results reported in the legend for Fig. 1. Importantly, GAPDH, tERK, and tAkt protein levels were not changed (see A; GAPDH and tAkt quantified data not shown). PAG (D) and caudal brain stem (E) ERK signaling changes are indicated. pERK was normalized to the tERK signal and further normalized to the vehicle:vehicle group. **, ***, and ****, p < 0.01, 0.001, and 0.0001, respectively, versus vehicle:vehicle group by Fisher's least significant difference post-hoc test. The reported sample size of individual mice for both D and E consisted of two technical replicates each performed by different experimenters at different institutions. Hsp90 inhibition increased the ERK signaling baseline, and stimulation above that baseline by DAMGO was completely lost in both brain regions. PAG (F) and caudal brain stem (G) Akt signaling changes are indicated. Akt was analyzed as described for ERK from the same samples and with the same technical replicates. * and **, p < 0.05 and 0.01 versus vehicle:vehicle group by Fisher's least significant difference post-hoc test. Hsp90 inhibition induced a very similar, albeit less robust pattern in Akt as in ERK. H, in a control experiment, ERK signaling was assessed in mice injected i.c.v. with the highest dose of 17-AAG tested in the dose response (5 nmol). PAG ERK signaling in these mice was assessed as described in D and E. *, p < 0.05 versus vehicle:vehicle group by Fisher's least significant difference post-hoc test. 5 nmol of 17-AAG induces the same ERK pattern as seen in D and E with no increased magnitude of the effect, suggesting that 0.5 nmol has a maximal effect on brain signaling, justifying the use of the lower dose. The reported sample size of individual mice consisted of one technical replicate.