SCAP is required for GPI-anchored protein transport from the TGN to the PM. (A) mKate2-FM4-GPI transport from the ER to the PM via the Golgi complex in control (Cont), SCAP, and VAP-A/B knockdown cells. 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 = 230–252 cells per condition). (B) mKate2-FM4-GPI has accumulated at the TGN at 90 min after the transport induction in SCAP knockdown cells. N, nucleus. (C) Colocalization of PAUF-MycHis and mKate2-FM4-GPI in CARTS at 30 min after the transport induction. (D) Colocalization of GST-PKD2-KD and mKate2-FM4-GPI in tubules attached to the TGN at 90 min after the transport induction. (C and D) High magnifications of the boxed areas are shown in the right column where brightness/contrast enhancement was applied to the mKate2-FM4-GPI channel. Arrowheads in D indicate a GST-PKD2-KD–induced tubule containing mKate2-FM4-GPI. (E) Colocalization of EQ-SM and PAUF-MycHis. High magnifications of the boxed areas are shown in the insets. The box-and-whisker plots show quantification of EQ-SM–positive puncta containing PAUF-MycHis (green) and PAUF-MycHis–positive puncta containing EQ-SM (magenta). Boxes delimit the first and third quartiles, and the central line is the median. The whiskers represent the minimum and maximum values after outlier removal (EQ-SM positive: n = 4,487 puncta; PAUF positive: n = 1,650 puncta in 12 cells; ****, P < 0.0001; paired two-tailed Student’s t test). Scale bars, 10 µm.