Ab52771

Reaction mixtures containing 1 μM purified human YAP protein (Abnova, Taiwan, # H00000116-P01), 0.125 μM human OGT (Abnova, H00008473-P01) and 10 μM UDP-GlcNAc (Sigma) in a buffer of 50 mM Tris/HCl (pH 7.5) and 1 mM DTT were incubated at 37 °C for 90 min. Then, co-IP was performed with anti-YAP antibodies (Abcam, #ab52771, 1:100) and detected by WB using anti-YAP (Santa Cruz, #sc-101199, 1:1000) and anti-O-GlcNAc (Abcam, #ab2739, 1:1000) antibodies.

After whole cell lysate (WCL) samples were acquired, cell lysates were incubated with protein A/G magnetic beads (Novex, Oslo, Norway) and western/IP lysis buffer (Beyotime, Haimen, China). Immunoprecipitates were washed five times. WCL samples were first subjected to IB analysis. The target proteins bound by the antibody were adjusted to the same content in each co-IP sample according to the gray value. Then, the co-IP samples were subjected to IB analysis to investigate the binding amount of other proteins to the target protein. The antibodies used for co-IP were anti-Myc (CST, #2276 or #2278), anti-HA (CST, #3724 or #2367), anti-YAP (Abcam, #ab52771 or Santa Cruz, #sc-101199), anti-TFCP2 (CST, #80784) and anti-KAT5 (Abcam, #ab137518).

Co-immunoprecipitation (co-IP) was performed as described previously [31 (link)]. After cells were harvested, the lysates were mixed with 50 ul of protein A/G-magnetic beads (Novex, Oslo, Norway) and incubated at 4 °C overnight with the indicated antibodies cross-linked to protein A/G magnetic beads. The beads were washed three times with Western/IP lysis buffer (Beyotime, Haimen, China), suspended in SDS-PAGE loading buffer, and then detected by IB with relevant antibodies. The antibodies used for co-IP were: anti-YAP (Abcam, #ab52771 and Santa Cruz, #sc-101199), anti-βTrCP (Abcam, #ab71753), anti-HA (Abcam #ab18181), anti-FLAG (CST, #2368), anti-CK1 (Abcam, #ab270997), anti-TEAD4 (Abcam, #ab197589) and anti-LATS1 (Abcam, #ab234820).