Anti mecp2 antibody

PC3 and MDA-MB-468 cells were cultured and seeded in p150 mm dishes at 37°C under atmospheric oxygen conditions. Once 70% confluent, cells were treated with DMSO, 2µM panobinostat, 10 µM Inhibitor-IV, 10 µM Inhibitor-VII, and 10 µM pracinostat for 45 min to 1.5 h and harvested in RIPA buffer (with complete protease inhibitor cocktail, 1 µM Trichostatin A and 1 mM nicotinamide). Protein concentration was quantified by the BCA method. Immunoprecipitation was performed using 4 μg of anti-MeCP2 antibody (Cell Signaling) and incubated for 2 h at 4°C. Protein A dynabeads (Invitrogen) were added to the immune-complex and incubated for 2 h at 4°C. IP protocol was followed as mentioned above. Beads were washed with RIPA buffer (four times) and autoclaved water (two times). Dry beads were shipped to Applied Biomics Inc. (Hayward, CA) for acetylation site identification by LC–MS/MS mass spectrometry on a fee-based service. The specific lysine residues that were acetylated, exhibited ion peaks at mass/charge (m/z) ratio of ~126 as summarized in Figure 4A.

Cells were plated at 30,000 cells per well in 8-well Lab-Tek II chamber slide (Thermo Fisher Scientific) 24 h prior to transduction. 24 h after plating, cells were transduced with AAVHSC7-226 at a MOI: 150,000. Before staining, cells were washed with PBS and fixed with 4% paraformaldehyde 48 h after transduction. They were then rinsed 3-times with PBS and blocked with PBS containing 3% BSA and 0.3% Triton X-100 for 1 h at room temperature. This was followed by incubation with primary anti-MeCP2 antibody (1: 100 dilution; Cell Signaling, #3456) and an anti-GFP antibody (1:500 dilution, Thermo Fisher, # MA5-15256) in PBS containing 1% BSA and 0.3% Triton X-100 at 4°C overnight. The cells were then rinsed 3-times with PBS and incubated with secondary antibodies. Secondary antibodies used were anti-rabbit Alexa Fluor-555 IgG (1:500, cell Signaling, #4413) and anti-mouse Alexa Fluor-488 IgG (1:500, cell Signaling, #4408) in PBS containing 1% BSA and 0.3% Triton X-100 for 2 h at room temperature in dark. The cells were finally rinsed 3-times with PBS, mounted with antifade reagent containing DAPI (VECTASHIELD Vibrance^®, #H-1800) and visualized at ×40 magnification using the Zeiss LSM 700 confocal microscope.