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. Author manuscript; available in PMC: 2014 Jun 19.
Published in final edited form as: Sci Signal. 2014 Jan 14;7(308):ra4. doi: 10.1126/scisignal.2004331

Figure 2. Acute haloperidol treatment leads to transient increases in protein synthesis that are mTORC1 dependent.

Figure 2

(A) Primary DIV7 striatal neurons were treated with haloperidol (Hal) or DMSO (Veh) for 30, 60 minutes, or 4 hours and 1 μg/ml puromycin. Puromycin was detected by Western blotting. A representative blot is shown. The 4-hr blot is a lighter exposure due to the intensity of the signal. Data were quantified as the percent of puromycin signal in the haloperidol-treated samples / puromycin signal in vehicle–treated samples normalized to actin and shown as the average (30 min; n=8, p =0.007; 60 min, n=7, p=0.005; 4 hours, n=4, not significant). Actin is a loading control. (B) The effect of S6K1 shRNA or the doxycycline-inducible (DOX) dominant-negative 4E-BP AA on haloperidol-mediated stimulation of translation. Data were quantified as in panel A for the 4E-BP AA “on” (Dox; n=4) and “off” (No Dox; n=3) conditions. * p=0.02). Doxycycline with and without S6K1 inhibitor (S6K1i), n = 3, not quantified due to comparatively light signal. HA indicates the presence of 4E-BP AA. Actin is a loading control. (C) The effect of S6K1 knockdown (S6K1 shRNA) or control (S6K1 scramble) on the haloperidol-induced increase in puromocyin incorporation. Data are quantified as in panel A. (n=4, p=0.03,). (D) Effectiveness of S6K1 knockdown was quantified for each experiment, knockdown average was 41% (n= 4 p < 0.0003). (E) Western blot analysis of S6 and clathrin abundance in striatal neurons exposed to vehicle or haloperidol under the same conditions used for the puromycin analysis (% Haloperidol/Vehicle normalized to actin loading control). (S6, n=3, p=0.003) (clathrin, n=4, p=not signficant). (F) Western blot analysis of eEF2 abundance in striatal neurons exposed to vehicle or haloperidol (20 μM, n=7, p=0.027), risperidone (Risperidone 100 nM, n=,5, p < 0.06), or amisulpride (Ami, 1 μM, n = 5, p = 0.08) for 4 hours. All graphs shown are average ± SEM and analyzed by Student’s t test. P < 0.05 was considered significant and is marked with asterisks; P< 0.1 is marked with #.