HERC3 directly interacts with the exposed CFTR-MSDs in vitro. (A) Western blotting confirmed the synthesis of FLAG-HERC3 (left) and biotinylated CFTR full-length (FL), M1, and M2 (right) using a wheat cell-free synthesis system in the presence or absence of asolectin liposomes. (B and C) AlphaScreen was employed to evaluate the direct binding of FLAG-HERC3 and biotinylated CFTR synthesized in the presence or absence of asolectin liposomes. FLAG- DHFR served as a negative control. The specific binding signal of FLAG-HERC3, subtracted by the DHFR binding, was measured in C. (D) The proposed model illustrates the function of HERC3 in the CFTR ERQC. HERC3 appears to selectively interact with specific regions of MSDs, typically embedded in the ER membrane. It is speculated that HERC3 monitors the MSDs of select membrane proteins at the ER membrane’s surface and facilitates the ERAD when the TM segments become exposed to the cytosol. Source data are available for this figure: SourceData F10.