, Cell Signaling Technologies), ERK1/2 (#9102, Cell Signaling Technologies), G?olf (produced as described 66 )
Cell Signaling Technology) mouse monoclonal antibodies: Pyk2 (#3480, Cell Signaling Technology), DARPP-32 (1:5000; mAb6, gift from Pr. Greengard) and phosphotyrosine, Cell Signaling Technology) chicken polyclonal antibody: tyrosine hydroxylase (#TH, Aves Labs, pp.5-947 ,
, IgG DyLight ? 800 or 680 conjugated antibodies (1:10000; Rockland Immunochemicals
For loading control a mouse monoclonal antibody for ?-actin was used (1:5000, Secondary antibody binding was detected by Odyssey infrared imaging apparatus, vol.5441 ,
, Mice were i.p. injected with cocaine (20 mg/kg) and placed in a 43 cm × 27 cm cage
, After 10 minutes, mice were euthanized and heads dipped in liquid nitrogen for 5 seconds. The striatum was dissected out, lysed by sonication in 250 ?l NP-40 lysis buffer [150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 10 mM NaF, 1% NP40 (v/v) supplemented with 1 mM sodium orthovanadate, phosphatase inhibitor
, Lysates were centrifuged for 20 min at 20,937 g (4 °C)
, GE Healthcare) and saturated with BSA (25 g/L). The beads were then mixed for 1 hour at 4 °C with rabbit polyclonal anti-Pyk2 antibody (1.7% v/v) (#P3902, Protein A-Sepharose beads (GE Healthcare) were pre-cleared by mixing with 14% Sephacryl S-100 (v/v)
If no difference from normality was detected, statistical analysis was performed using two-tailed Student's t test or ANOVA and Sidak's post-hoc test. Otherwise or when n was <7, non-parametric two-tailed Mann-Whitney test was used ,
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