Skip to main content
. 2017 Apr 27;292(25):10414–10428. doi: 10.1074/jbc.M116.769489

Figure 1.

Figure 1.

Hsp90 inhibition has multivariate effects on protein expression and signal transduction in vitro. Four different cell models of MOR signal transduction were treated with 1 μm 17-AAG or vehicle for 24 h, serum-starved (see “Experimental procedures”), and treated with vehicle, 10 μm DAMGO, or 10 μm LPA (a non-opioid GPCR endogenous agonist) for 10 min. Results were measured by Western blot, and analyzed as reported under “Experimental procedures.” The sample size reported in each experiment graph represents an independent technical replicate consisting of two or more averaged replicate wells counted as n = 1. All data are reported as the mean ± S.D. A, representative Western blots for each target are shown, with vehicle (Veh) or 17-AAG (AAG) treatment. The cell line from which each representative blot was taken is indicated below the target name. The numbers with arrows on the left side of each image represents the position of the nearest molecular weight markers. These images also indicate that GAPDH and tERK were not altered by 17-AAG treatment (data not shown). B, the expression levels of four separate signaling proteins normalized to GAPDH and vehicle control were analyzed in each cell line as labeled. *, ***, and ****, p < 0.05, 0.001, and 0.0001, respectively, versus same cell line vehicle control by unpaired 2-tailed t test. Only Hsp70 shows consistent regulation across cell lines with Hsp90 inhibition. C, signal transduction via ERK phosphorylation is reported for each cell line in a separate graph. The pERK signal was normalized to tERK and, in the case of CHO, was normalized further to MOR levels as they were decreased. The pERK/tERK signal was further normalized to the vehicle:vehicle group. * and **, p < 0.05 and 0.01 versus same pretreatment group (vehicle, 17-AAG) vehicle control; #, ##, ###, and ####, p < 0.05, 0.01, 0.001, and 0.0001, respectively, versus vehicle:vehicle group; both sets by two-way ANOVA with Fisher's least significant difference post-hoc test. Each line showed a different alteration in the ERK signaling pattern after Hsp90 inhibition, and both DAMGO and LPA showed similar behavior.