Binding assays

Lysates (100 μg/sample) were incubated with 10 μg of an anti-MAPKAP kinase-2 antibody on a rocking platform at 4℃. After 3 h, 50 μl of a protein A/G-agarose slurry was added, and tubes were rocked for another 1 h at 4℃. Agarose beads were pelleted by centrifugation at 1500 × g for 5 min., washed three times with lysis buffer and washed once with 20 mM Hepes, pH 7.0 buffer. Immunoprecipitations were resuspended in 75 μl of kinase assay buffer (20 mM Hepes, pH 7.0, 5 mM 2-mercaptoethanol, 10 mM MgCl2, 0.1 mg/ml bovine serum albumin, containing 2 μg of hsp27. Kinase reactions were initiated by the addition of 10 μM ATP plus 10 μCi of [γ-33P]ATP and incubated at 25℃ for 30 min. Reactions were terminated by the addition of Laemmli SDS sample buffer, boiled for 5 min, electrophoresed on a 12% Tris-glycine gel, dried, and quantitated using a Molecular Dynamics phosphorimager.

Animal models

Adult male CD-1 mice, Morphine-pretreated rats

Preparation method

The solubility of this compound in DMSO is >16.8mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Dosage form

50 mg/kg, diluted 1:1 in water and DMSO immediately before the injection, Intraperitoneal injection,

Application

In adult male CD-1 mice, SL-327 (50 mg/kg, i.p.) inhibited Pp-ERK immunostaining in the nuclei of the cells induced by cocaine. SL-327 (50 mg/kg, i.p.) pretreatment inhibited c-Fos expression in nuclear and inhibited activation of ERK within all the amygdala. In morphine-pretreated rats, SL-327 (20 mg/kg, i.p.) increased (58%) the expression of morphine-induced psychomotor sensitization (SW3) and fully prevented the upregulation of p-PEA-15, p-FADD, and p-Akt1 at SW3.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

SL-327 is a selective inhibitor of MEK1 and MEK2 with IC50 values of 0.18 and 0.22μM,  respectively.
MEK1 and MEK2 (ERK) are a kinase enzyme which phosphorylate mitogen-activated protein kinase (MAPK). Extracellular signal-regulated kinase (ERK) activity is essential for the acquisition of associative learning tasks.
In adult male CD-1 mice, SL-327 inhibited Pp-ERK immunostaining in the nuclei of the cells induced by cocaine. SL-327 pretreatment inhibited c-Fos expression in nuclear and inhibited activation of ERK within all the amygdala, namely LA, BLA, BMP, Ce and MePD [1]. In morphine-pretreated rats, SL-327 increased (58%) the expression of morphine-induced psychomotor sensitization (SW3) and fully prevented the upregulation of p-PEA-15, p-FADD, and p-Akt1 at SW3 [2]. In adult male DBA/2J mice, SL-327 significantly reduced pERK levels by 40% in both the motor cortex and dorsal striatum [3]. In rat model, SL-327 inhibited MAPK/ERK cascade, which prevented LTP-dependent gene induction and CREB and Elk-1 phosphorylation, resulting in rapidly decaying LTP [4].
References:
[1]. Radwanska K, Caboche J, Kaczmarek L. Extracellular signal-regulated kinases (ERKs) modulate cocaine-induced gene expression in the mouse amygdala. Eur J Neurosci, 2005, 22(4): 939-948.
[2]. Ramos-Miguel A, Esteban S, García-Sevilla JA. The time course of unconditioned morphine-induced psychomotor sensitization mirrors the phosphorylation of FADD and MEK/ERK in rat striatum: role of PEA-15 as a FADD-ERK binding partner in striatal plasticity. Eur Neuropsychopharmacol, 2010, 20(1): 49-64.
[3]. Groblewski PA, Franken FH, Cunningham CL. Inhibition of extracellular signal-regulated kinase (ERK) activity with SL327 does not prevent acquisition, expression, and extinction of ethanol-seeking behavior in mice. Behav Brain Res, 2011, 217(2): 399-407.
[4]. Davis S, Vanhoutte P, Pages C, et al. The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J Neurosci, 2000, 20(12): 4563-4572.