Antibody conjugated protein a g magnetic beads

For Co-IP, cells were lysed in 200 µl Western/IP lysis buffer (Beyotime Institute of Biotechnology) incubated with primary antibodies at 4°C overnight. Subsequently, cell lysates (0.4 ml) were incubated with antibody-conjugated protein A/G magnetic beads (30 µl, Thermo Fisher Scientific, Inc.) at 4°C for 3 h. After centrifugation (800 × g) at 4°C for 5 min, immunoprecipitate isolated with magnetic beads were washed five times with Western/IP lysis buffer and boiled with sample loading buffer. The resulting immunoprecipitate was analyzed using western blotting, as aforementioned. The primary antibodies used for Co-IP were as follows: OTX1 (1:1,500; cat. no. sc-517000, Santa Cruz Biotechnology, Inc.), Wnt9b (1:1,000; cat. no. abx178928; CiteAb, Ltd.) and GAPDH (1:1,000; cat. no. ab181602; Abcam). The secondary antibody used for Co-IP was Goat anti-Rabbit IgG (H + L) secondary antibody (1:500; cat. no. 31340; Thermo Fisher Scientific, Inc.).

For co-IP, cell lysates (containing 2×10⁷ cells) were incubated with antibody-conjugated protein A/G magnetic beads (Thermo Fisher Scientific, Inc.) in Western/IP lysis buffer (Beyotime Institute of Biotechnology) at 4°C overnight. Immunoprecipitates isolated with the magnetic beads were washed five times with Western/IP lysis buffer before being subjected to IB. The antibodies used for co-IP were: Anti-Myc (product no. 2276; 1:100; CST), anti-HA (product no. 2367; CST, 1:100), anti-CREB (product no. 9104; 1:100; CST), anti-EP300 (product code ab54984; 1:100; Abcam) and anti-SETDB2 (product code ab13712; 1:50; Abcam).