Cab45 binds secretory cargo. (A) GST and GST Cab45 expressed in E. coli were purified on GST beads. The respective preparations of beads were incubated with HeLa cell lysate for 4 h at 4°C, washed extensively, and finally incubated with SDS-PAGE sample buffer to release the bound proteins, which were then analyzed by SDS-PAGE. Gels were stained with Coomassie blue, and protein bands were excised and analyzed by mass spectrometry. The full list of identified proteins is shown in Table S1. This pool contained several proteins that are secreted or transported to the plasma membranes and are shown here. (B) Schematic presentation of Cab45 wt and the amino acids mutated in the EF hand domains of Cab45 mutant. (C) Recombinant purified GST-Cab45 wt or GST-Cab45 mutant bound to glutathione Sepharose beads were incubated with HeLa cell lysates. Beads were washed extensively and then incubated with SDS-PAGE sample buffer to elute the bound proteins, which were Western blotted with antibodies to Cathepsin D, COMP, MGP, and GST. (D) Cells expressing Flag-LysC were lysed and incubated with anti-Flag beads for 1 h at 4°C. Beads were then incubated with recombinant GST-Cab45 wt or GST Cab45 mutant. Bound proteins were eluted with SDS-PAGE sample buffer and Western blotted with anti-GST and anti-Flag antibodies. (E) HeLa cells stably expressing HA-Cab45 wt were incubated with PBS-DMSO or 1 mM PBS-DSP for 30 min at room temperature. The reaction was terminated and the HA-tagged Cab45 was immunoprecipitated and analyzed by Western blotting with antibodies against HA and COMP.