Antirabbit gfp

HEK293T cells were grown in Dulbeco’s modified Eagle’s medium (Gibco™) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco™) and 1% Antibiotic-Antimycotic (Gibco™), and were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were transfected with constructs of mCherry-Fzd6 and GFP-VANGL1 (WT or mutants) using Lipofectamine2000 and cultured for 48 h. Cells were rinsed with cold PBS twice and then lysed with 1 × NP40 Lysis buffer (Invitrogen™) with cOmplete™ ULTRA Tablets (Millipore Sigma) for 20 min. The protein lysates were immunoblotted with antirabbit-GFP, anti-mCherry, and anti-rabbit-GAPDH (1:1000, Cell Signaling, Danvers, MA, 2218S) overnight. IRDye® 800CW goat anti-rabbit IgG secondary antibodies (LI-COR, Cambridge, UK) were cultured for 1 h. The images were captured using Odyssey^® (LI-COR). The statistical analyses were performed using a Student t test on data obtained from three independent experiments.

For cell fractionations, cells were suspended in fractionation buffer (20 mM HEPES, 10 mM KCl, 150 mM sucrose, 2 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1mM DTT, protease/phasphatase inhibitor, pH 7.4) and lysed with brief sonication (2 times, 3 s, 10% amplitude). Lysate was centrifuged at 3500 rpm (Sorvall™ Legend™ Micro 17 microcentrifuge, Thermo Fisher Scientific, Waltham, MA) for 10 min at 4 °C to remove cell debris and nucleus. Supernatant (300 μg protein/100 μl) was centrifuged again at 40,000 rpm for 2 h at 4 °C using Optima XE ultracentrifuge (Beckman Coulter, Indianapolis, IN). This supernatant was cytosolic fraction. The pellet (membrane fraction) was incubated in fractionation buffer containing 1% Triton X-100 overnight at 4°C, and then homogenized by brief sonication. The cytosolic and membrane fractions were precipitated by trichloroacetic acid (20%). The pellets were washed twice with 70% acetone, and re-dissolved in 40 μl of Laemmli sample buffer [51 (link)]. Cell fractions were subjected to SDS-PAGE and immunoblot analysis as described earlier [52 (link)]. The anti-rabbit GFP (1:1000); anti-rabbit β-actin (1:2000); and anti-rabbit HRP-conjugated (1:5000) (Cell Signaling Technology, Inc. Danvers, MA) were used for immunoblot analysis.