Association of p23 and p24 with remodeled GPI-APs. (A) Detection of proteins specifically associated with VFG-GPI in FF8. VFG-GPI was expressed and accumulated in the ER in FF8, FPRC2, and C19 cells. The cells were then incubated at 32°C for 20 min to initiate transport of VFG-GPI. After cell lysis, VFG-GPI was precipitated with anti-Flag beads. Co-precipitated proteins were eluted using the Flag peptide and subjected to SDS-PAGE and silver staining. The boxed area at ∼20 kD is enlarged to the right. (B) Sequences of hamster Tmed10 (p23) and Tmed2 (p24). Protein bands at 20 kD in A were digested in-gel with trypsin and analyzed by mass spectrometry. The fragments detected by MS/MS analysis are shown in red. (C) Precipitated proteins in A were analyzed by Western blotting using a rabbit anti-p23, anti-p24, or anti-ERGIC53 polyclonal antibody. VFG-GPI was detected with an anti-GFP antibody. (D and E) Immunoprecipitation of VFG-GPI with p23 and p24. FF8, FPRC2, and FPRC2 stably expressing PGAP1 (D) or FF8, C19, and C19 stably expressing HA-PGAP5 (E) were cultured with 1 µg/ml doxycycline at 40°C for 24 h to induce VFG-GPI expression and its accumulation in the ER. The cells were then incubated at 32°C for 20 min to initiate VFG-GPI transport. After cell lysis, VFG-GPI was precipitated with anti-Flag beads and coprecipitated proteins were detected by immunoblotting using an anti-p23, anti-p24, or anti-ERGIC53 antibody. VFG-GPI was detected with an anti-GFP antibody. Total lysate corresponding to 1% and immunoprecipitates were used for analysis.