Lumi light western blotting substrate kit

Cell lysates were prepared by washing cells with ice-cold PBS twice, and then lysing on ice for 5 min with RIPA. Samples were assayed for protein concentration using Coomassie (Bradford) Protein Assay Kit (Thermo Scientific), and then mixed with 6x SDS sample buffer before resolving by 6%-10% SDS-PAGE, transferring onto PVDF membrane, and immunoblotting. For immunoblotting, membrane was incubated with anti-MeCP2 (1:1000, Abcam, Sigma Chemical Co.), anti-non-phosphorylated (S33/37/T41) β-catenin (1:1000, Cell Signaling Technology), and anti-β-actin (1:5000, Santa Cruz Biotechnology) antibodies in 5% non-fat-milk/1x TBS containing 0.1% Tween20 overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Proteins were detected by a chemiluminescent method using Lumi-Light Western Blotting Substrate kit (Roche), and/or SuperSignal West Femto kit (Pierce). Densimetry of detected bands for protein of interest was performed by using Bio-Rad ChemiDoc^TM MP Imaging System and the data were standardized by β-actin density.

Cells were grown to 80% confluency, washed with PBS and lysed for 30 minutes on ice (Tris-HCL 50mM, NaCl 100 mM, Triton-X 1%, deoxycholute 0.5%, MgCl2 10mM, NaVO3 1mM, NaF 50mM, PMSF 1mM, protease inhibitor in PBS). SDS-PAGE analysis was performed, and membranes were incubated overnight at 4° with antibodies directed against the indicated proteins. Membranes were washed, incubated with the appropriate secondary antibodies and exposed to Lumi-Light Western Blotting Substrate kit (Roche Diagnostics Corporation, Indianapolis, IN). Images were analyzed with ImagePro (MediaCybernetics, Bethesda, MD) and Prism (GraphPad Software, La Jolla). Densitometry data were analyzed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon modified Newman-Keuls-Student procedure, where appropriate. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant and all are two-tailed.