Figure S1.

Cargo secretion in SEC24C KO, NanoBiT mutant stability and SURF4 mutagenesis. (A) Radiolabeled pulse-chase experiment testing Cab45 secretion in WT and SEC24 KO cells. Transiently transfected HA-tagged Cab45 was immunoprecipitated from media and lysates at indicated time points after [³⁵S]-Met/Cys addition and detected by SDS-PAGE and phosphorimage analysis. n = 1. (B) Stability of indicated SEC24 constructs was tested in appropriate KO cells by immunoblotting for steady-state protein levels using the antibodies indicated. β-actin served as loading control. (C) SURF4 topology prediction based on trRosetta, TOPCONS, and other topology prediction algorithms (see Materials and methods), with conservation and disease variants mapped from gnomAD. Based on this prediction, mutants in cytosolic or lumenal regions were generated. Those labeled in green indicate defects in Cab45 secretion but stable expression of the mutant. (D) Immunoblots of steady-state FLAG-SURF4 and secreted Cab45 upon transient expression of FLAG-SURF4 WT and mutant constructs in HEK-293TREx SURF4 KO. Actin serves as a loading control for lysates. Mutants in green had reduced or no Cab45 secretion rescue in the media. (E) Stability of indicated SURF4 constructs was tested in appropriate KO cells by immunoblotting for steady-state protein levels using the antibodies indicated. β-actin served as a loading control. Source data are available for this figure: SourceData FS1.