PCAF interacts with XBP-1S and mediates XBP-1S-dependent transcription

Lew, Qiao Jing; Chu, Kai Ling; Lee, Jialing; Koh, Poh Ling; Rajasegaran, Vikneswari; Teo, Jin Yuan; Chao, Sheng-Hao

Domain study of XBP-1 was carried out using a series of HA-tagged XBP-1 truncations (Figure 2A ). Cells were transfected with an individual XBP-1 truncation plasmid followed by IP using anti-PCAF antibodies. As shown in Figure 2B , only the XBP-1S-specific C-terminal region, which contained the transcriptional activation domain of XBP-1S, was found to associate with PCAF, but not the XBP-1U-specific C-terminus or any other regions of XBP-1. The heavy chains of anti-PCAF antibodies were also recognized by the secondary antibody used for the immunoblotting. Since the molecular weights of heavy chains and HA-tagged XBP-1S were similar (50 kDa), the blot could not reveal the presence of HA-XBP-1S in the immunoprecipitates. We did another western blot using an anti-XBP-1S antibody recognizing the common domain of XBP-1S and XBP-1U and confirmed the interaction between PCAF and HA-XBP-1S ( Figure 2B , the anti-XBP-1 blot). It was noted that the interaction between PCAF and endogenous XBP-1S proteins were also detected in the HA-1S(134-376)-and HA-XBP-1U-transfected cells ( Figure 2B) . Collectively, the results demonstrate that PCAF binds to XBP-1S through the transcriptional activation domain of XBP-1S located in its C-terminal region.