p24 proteins associated with GPI-APs in a pH-dependent manner. (A) Immunoprecipitation of VFG-GPI with p23 and p24 at various pH levels. FF8 cells were cultured with 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 in lysis-IP buffer III of the indicated pH, VFG-GPI was precipitated with anti-Flag beads and washed five times in wash buffer III of the indicated pH, followed 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. Bands in 1% total fractions gradually increased with an increased pH partly because solubilization of proteins in 1% digitonin was slightly better at higher pH. (B) Release of p23 and p24 from VFG-GPI by lowering pH. FF8 cells prepared in a similar manner to A were lysed in lysis-IP buffer III, pH 7.4, and VFG-GPI was precipitated with anti-Flag beads. After washing five times with wash buffer III, pH 7.4, wash buffer III at the indicated pH was added and incubated at 4°C for 15 min. The supernatant (S) and bead pellets (P) were collected, followed by immunoblotting as in A.