Identification of potential CCDC134 interactors by immunoprecipitation and LC-MS analysis

Our data indicate that CCDC134 plays a critical role in regulating the biogenesis of Toll-like receptors (TLRs). To gain deeper insights into the molecular mechanisms through which CCDC134 mediates TLR regulation, we sought to identify potential binding partners using interaction proteomics. Mass spectrometry analysis of Flag-CCDC134 immunoprecipitates from stably reconstituted CCDC134 knockout CAL-1 cells provided a better understanding of the interaction landscape associated with CCDC134. Notably, among the identified proteins, the TLR chaperone Gp96 emerged as one of the strongest binding partners. We further validated that CCDC134 interacts with Gp96 and is essential for its proper folding and stability. Consequently, loss of CCDC134 impairs the folding, maturation, and trafficking of TLRs.