P1149

Hippocampal cultured neurons were prepared from rat embryos at embryonic day 18 (E18; n = 15 pregnant females) as described previously (Zhang and Macara 2006 (link)) with minor modifications. Cells were dissociated mechanically after trypsin treatment and plated onto poly-D-lysine-coated (Sigma, P1149) plastic culture dishes at a density of ∼5 × 10⁴ cells per square centimeter. Plated cells were recovered in the plating medium (DMEM with 2 mM glutamine, 10% FBS, 0.45% glucose, 0.11 mg/mL sodium pyruvate) for 3–4 h and then maintained in the neurobasal medium (Invitrogen, 21103-049) supplemented with B27 (Invitrogen, 17504-044) and glutamax (Invitrogen, 35050-061) before use.

COS-7 cells were seeded at 5 × 10⁴ cells/well in 24-well plates coated with poly-D-lysine (0.1 mg/ml; P1149-Sigma) and incubated in 5% CO₂ at 37 °C overnight. Cells were transfected with myc-α1, β3, and γ2 cDNA or α1, myc-β3, and γ2 cDNA (wt or mutated) using Lipofectamine LTX reagent. After transfection and treatment (if applicable), cells were washed twice with PBS and fixed for 10 min with 4% PFA/sucrose in PBS. Cells were rinsed twice with PBS and washed twice with Hank’s Balanced Salt Solution (HBSS; 14185–052-Gibco) for 10 min. Nonspecific binding of primary antibodies was blocked by application of rabbit serum (10%)/BSA (1%) in HBSS for 30 min at room temperature. For total levels, cell membrane was permeabilized by addition of Triton X-100 (0.5%). Cells were incubated with mouse anti-myc antibody (2 μg/ml; M4439-Sigma) in BSA (1%)/HBSS at 4 °C overnight. Cells were washed with HBSS, blocked as above and incubated with HRP-conjugated rabbit anti-mouse IgG (0.4 μg/ml; 61-6520-Invitrogen) in BSA (1%)/HBSS for 2 h at room temperature. Cells were rinsed twice and washed twice for 20 min with HBSS. HRP-substrate 3,3′,5,5′-tetramethylbenzidine (0.5 ml; T0440-Sigma) was applied to each well at 5-s intervals. Reaction was stopped by transferring reagent to 1.5 ml water. Absorbance was measured at 655 nm in a Beckman DU800 spectrophotometer.