p24 family is required for the GPI-AP transport from the ER. (A) Relative amount of p23 and p24 mRNA from FF8 cells stably transfected with empty vector (+Vec) or p23 siRNA vector (+242) as determined by quantitative RT-PCR analysis. (B) Western blotting of p23 and p24 in p23 knockdown cells. FF8 cells permanently transfected with empty vector (Vec) or p23 siRNA vector (242) were lysed and the proteins were resolved by SDS-PAGE, followed by immunoblotting using rabbit anti-p23, anti-p24, or anti-ERGIC53 polyclonal antibody. (C) The cell surface expression of VFG-GPI was traced by flow cytometry. FF8 cells stably transfected with empty vector (+Vec) or p23 siRNA vector (+242) were stained with anti-Flag antibody at the indicated times after the commencement of transport. The geometric mean fluorescent values of surface VFG-GPI were quantified at each time point. The geometric mean of FF8 + Vec at 60 min was plotted as 100% relative transport. Values represent the mean ± SD (error bars; n = 3). (D and E) Pulse-chase transport analysis of Venus-CD59. Cells stably expressing Venus-CD59 were transfected with siRNAs against p23 or control RNA. After 72 h, the cells were pulse-labeled with [³⁵S]methionine/cysteine followed by chasing for the indicated times, and immunoprecipitated with an antibody against Venus. Immunoprecipitates were treated with Endo-H, separated by SDS-PAGE, and analyzed and quantified using a Cyclone Phosphor Imager (Packard). The proportion of Golgi form (Endo-H–resistant form) and Venus-CD59 were plotted in E.