Immunoprecipitation and Immunoblotting for GATA1

Before the collection of protein extracts, cells were dissolved in lysis buffer. The cell lysis buffer was mixed with Flag M2-affinity gel-conjugated GATA1 antibody at 4°C for 12 h. After that, the protein was gradually eluted by KCl-contained buffer. The lysates (approximately 300 µg protein) were incubated with fresh protein A-beads (35 µL, #9863, CST) and 1 µg anti-GATA1 (1:1,000, ab181544, Abcam), anti-Flag (1:500, AF519, Beyotime) or IgG (1:500, ab172730, Abcam) at 4°C for 3 h. The magnetic beads were lysed in lysis buffer and loaded in SDS-PAGE. The immunoprecipitated proteins were reacted with anti-HDAC1 (1:1,000, ab109411, Abcam), anti-HDAC2 (1:1,200, ab32117, Abcam) and anti-HDAC3 (1:2,000, ab32369, Abcam). The immunoblot reactions were performed as described previously [25 (link)].