Ni nta his binding resin

As described previously52 (link), cultured mouse cortical neurons were collected and lysed in RIPA buffer and MeCP2 antibody (Cell signaling technology, Beverly, MA, USA) was used for protein immunoprecipitation and normal rabbit IgG (Santa Cruz, CA, USA) was used as negative control.
Immunoprecipitation in rat cortex was performed as following: Mecp2-null rat cortex and WT littermate cortex were collected and lysed with RIPA buffer, and were purified by Ni-NTA His binding resin (Millipore, Bedford, MA, USA) due to the tandem Histidine residues in the MeCP2 protein.

MeCP2 protein complexes were purified as described previously52 (link). Briefly, His-MeCP2 expression plasmid was transfected into 293 T cells and purified by Ni-NTA His binding resin (Millipore, Bedford, MA, USA) was used to purify overexpressed His-MeCP2 in 293-T cells, and after washing extensively for four times with Urea buffer, mass spectrometry was performed to identify the proteins closely associated with MeCP2.

Recombinant p24 proteins were purified from E. coli as previously described (59 (link)) with minor modification. For the expression and purification of the fusion protein, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET23a-p24. Protein expression was induced by adding 0.4 mM isopropyl β-d-thiogalactoside (Duchefa Biochemie, Haarlem, Netherlands). Bacterial cells were harvested and disrupted by sonication on ice for 10 min. Sonicated lysates were centrifuged at 1,600 × g for 20 min at 4°C, and the pellets containing p24 protein were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). The proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea, and residual salts. Purity of p24 protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel). The gel was visualized using Coomassie brilliant blue staining methods (60 (link)) (Figure S9 in Supplementary Material).