Western blotting

To examine the effect of FLX on MeCP2 levels, brain areas of Mecp2 HET and WT mice were dissected out free-hand. Striatum, hippocampus and cortex were homogenized in 1% Sodium dodecyl sulphate (SDS) and total protein concentration was determined by the bicinchoninic acid protein assay (BCA Protein Assay Kit, Pierce Biotechnology, USA). Twenty µg protein/sample were loaded onto 10% acrylamide gels. The amount of protein was established by running increasing amount of protein on the gel (Supplementary Fig. ⁴). For the MW estimates of target proteins, Precision Plus Protein Dual Color Standards (BIO-RAD, Cat. #1610374) was loaded in one well of each gel. Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham, GE Healthcare).

Membranes were cut approximately in correspondence to the 50 kDa MW. Blots were blocked with 5% non-fat milk in tris-buffered saline (TBST) (20 mM Tris, pH 7.6, 140 mM NaCl, and 0.5% Tween-20) for 1 h at room temperature. Membranes containing proteins heavier than 50 kDa were incubated overnight at 4 °C with primary antibody against MeCP2 (MW = 75 kDa) diluted 1:2000 (Cell Signaling Technology, Cat. # 3456), which detects both isoforms 1 and 2 of the protein (datasheet). Membranes containing proteins lighter than 50 kDa were incubated with the antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; MW = 37 kDa) (Abcam, Cat. # Ab22555) diluted 1:20,000, as a loading control. After washing with TBST (5 × 5 min), anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (Cell Signaling Technology, Cat. # 7074) were applied for 1 h at room temperature (1:5000 and 1:30,000 for MeCP2 and GAPDH, respectively). Primary antibodies were diluted in bovine serum albumin (BSA) 2% in TBST while the secondary antibody was diluted in 5% non-fat milk. The immune-positive protein bands were detected with a chemiluminescent home-made enhanced chemiluminescence (ECL) luminol/p-coumaric acid solution. Membranes were exposed to autoradiography films in the dark room (Hyperfilm ECL, Amersham GE Healthcare) then developed. Autoradiographs were scanned and signal intensity assessed with ImageJ (NIH) software. The ratio between MeCP2 and GAPDH for each sample was normalized to the WT mice average.