Rabbit polyclonal acetylated lysine antibody

Nuclear extracts were prepared from HEK293T cells, and immunoprecipitation studies and Western blotting were performed as previously described 75 (link). For the immunoprecipitation studies, an ANTI-FLAG® M2 Agarose Affinity Gel (Sigma-Aldrich, St. Louis, Missouri, United States), Anti-HA-tag pAb-Agarose (Medical & Biological Laboratories, Tokyo, Japan), anti-HA-tag mAb (Medical & Biological Laboratories), and normal murine IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) were used. Elution of the proteins was carried out with 100 μg / ml FLAG peptide (Sigma-Aldrich) or 0.1 M Glycine pH 2.5.
For Western blotting, rabbit polyclonal acetylated lysine antibody, (Cell Signaling Technology, Danvers, Massachusetts, United States), goat anti-GATA4 polyclonal antibody, murine anti-GATA4 monoclonal antibody, rabbit anti-p300 polyclonal antibody, murine anti-p300 monoclonal antibody, rabbit anti-V5-probe polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA), murine monoclonal FLAG (M5) antibody (Sigma-Aldrich), murine anti-FLAG monoclonal antibody, murine anti-HA monoclonal antibody, and murine anti-Myc monoclonal antibody (Medical & Biological Laboratories) were used. The levels of the bands were detected with an Amersham Imager 680 (Cytiva) and quantified using ImageJ software, version 1.52q.

Lysates from each group were generated with the Piece Co-IP Kit (Thermo Scientific, Bonn, Germany). Then the co-immunoprecipitation (Co-IP) was carried out according to the manufacturer's instructions. Briefly, the rabbit polyclonal β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA) was first immobilized for 2 h onto Amino Link Plus coupling resin. The resin was then washed and incubated with lysate overnight at 4°C. Subsequently, the resin was washed again and then eluted by elution buffer. Elution buffers were collected and analyzed by Western blotting which used rabbit polyclonal β-catenin antibody(Cell Signaling Technology, Danvers, MA,USA), rabbit polyclonal p300 antibody antibody (Santa Cruz Biotechnology), rabbit polyclonal acetylated-lysine antibody(Cell Signaling Technology) and peroxidase-conjugated goat anti-rabbit IgG (Bioss). A negative control was employed in the Co-IP kit to assess non-specific binding.