SL327 

Protein kinase assays are performed. All kinase assays are started by adding enzyme to a mixture that included [γ-³²P]ATP and substrate. This mixture is then incubated at 30°C or 37°C for 10 min. The reaction is stopped by spotting aliquots of the reaction mixture onto Whatman P-81 phosphocellulose filter paper. The papers are then washed in 150 mM H₃PO₄, dried, and subjected to scintillation counting. The catalytic subunit of PKA is assayed by measuring [³²P]phosphate incorporation into the substrate Kemptide (100 μM). The activity of CaMKII is determined by measuring phosphorylation of the synthetic peptide Autocamtide (100 μM) in the presence of 100 μM Calcium and 10 μg/mL Calmodulin. A synthetic peptide analog of a fragment of neurogranin, NG_(28-43) (10 μM) is used as a specific substrate for the catalytic subunit of PKC. In all cases, substrate phosphorylation was linear with respect to time and enzyme concentration^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[2]
Adult male 129S3/SvImJ mice are used. In the 1×-pairing paradigm of cue and contextual fear conditioning, animals are placed in the fear conditioning apparatus for 3 min, then a 30-sec acoustic conditioned stimulus (CS; white noise, 80 dB) is delivered. During the last second of the CS, a 1-sec shock unconditioned stimulus (US; 0.5 mA) is applied to the grid floor. To assess contextual learning, the animals are returned to the training context 24 hr post-training, and freezing behavior is scored for 5 min. To assess cue learning, the animals are placed in a different context (novel odor, lighting, cage floor, and visual cues) following contextual testing. Baseline behavior is measured for 3 min in the novel context (Pre-CS), then the tone is presented for 3 min. Freezing behavior is assessed with a time sampling procedure whereby the animal was observed for ~1 sec every 5 sec. The experimenter is blind to drug treatment. Animals are injected with either vehicle (2 mL/kg, 100% DMSO) or SL327 (10, 30, and 50 mg/kg; at 2 mL/kg, dissolved in 100% DMSO) intraperitoneally 1 hr before training^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Cheng Y, et al. Current Development Status of MEK Inhibitors. Molecules. 2017 Sep 26;22(10). pii: E1551.  [Content Brief]

  [2]. Selcher JC, et al. A necessity for MAP kinase activation in mammalian spatial learning. Learn Mem. 1999 Sep-Oct;6(5):478-90.  [Content Brief]