Effects of knockdown of OSBP, CERT/OSBP , or SCAP on trafficking of GPI-anchored protein or VSV-G. (A) mKate2-FM4-GPI transport from the ER to the PM via the Golgi complex in control (Cont), OSBP, and CERT/OSBP knockdown cells. The cells were incubated at 37°C with D/D solubilizer and cycloheximide and fixed at the indicated times. The graph shows the percentages of cells with mKate2-FM4-GPI at the ER, ER/Golgi, Golgi/PM, or PM at the indicated times. The data shown are for a single representative experiment out of three performed (n = 229–268 cells per condition). (B) mKate2-FM4-GPI transport in the presence or absence of the Myc-OSBP PH-FFAT mutant. Asterisks denote cells coexpressing mKate2-FM4-GPI and Myc-OSBP PH-FFAT. (C) VSV-G–GFP transport from the ER to the PM via the Golgi complex in control and SCAP knockdown cells. The cells were incubated at 32°C with cycloheximide and fixed at the indicated times. The graph shows the percentages of cells with VSV-G–GFP at the ER, ER/Golgi, Golgi/PM, or PM at the indicated times. The data shown are for a single representative experiment out of three performed (n = 203–224 cells per condition). Scale bars, 10 µm.