p24 proteins form heteromeric complexes with subfamily members. (A and B) Identification of proteins associated with VFG-GPI depending on pH. FF8 (WT) and FPRC2 (pgap1) cells were cultured with doxycycline at 40°C for 24 h. The cells were then incubated at 32°C for 20 min. After cell lysis using lysis-IP buffer II, pH 8.0, VFG-GPI was purified using an anti-Flag column. After thorough washing in wash buffer II, pH 8.0, the binding proteins were eluted with elution buffer II, pH 6.0. After elution with six bed volumes of buffer, proteins bound to the column were extracted with SDS sample buffer. Each fraction was subjected to SDS-PAGE, followed by immunoblotting using an anti-GFP or anti-p23 antibody (A). Proteins in eluted fraction 2 from FF8 (WT) and FPRC2 (pgap1) were detected by silver staining (B). Protein bands at 20 and 25 kD were identified by mass spectrometry as Tmed10 (p23), Tmed2 (p24), Tmed9 (p25), and Tmed5 (p28). Detected fragments are shown in Fig. S4. Protein bands indicated by an asterisk (*) were observed through all fractions (elutions 1−6) at similar levels and were not specific in WT cells. (C) Knockdown of p23 destabilized other p24 proteins. FF8 cells permanently transfected with an empty vector (Control) or p23 siRNA vector (p23KD) were lysed, and proteins were resolved by SDS-PAGE, followed by immunoblotting using rabbit anti-p23, anti-p24, anti-p25, anti-p28, and anti-ERGIC53 polyclonal antibodies. (D) Coimmunoprecipitation of myc-p23 with p24 proteins. FF8 cells were stably transfected with a retrovirus vector expressing RNAi-resistant myc-tagged p23 (myc-p23) and shRNA against endogenous p23, as described in Fig. S5. After cell lysis, myc-p23 was precipitated with anti-HA (control) or anti-myc antibodies or without antibody (No Ab), and coprecipitated proteins were detected by immunoblotting against anti-p23, anti-p24, anti-p25, anti-p28, and anti-ERGIC53 antibodies. Total lysate corresponding to 4% and immunoprecipitates were used for analysis. *, IgG heavy chains.