Figure 6.
SCAP is required for the biogenesis of CARTS at the TGN.(A) Close proximity of CARTS formation sites (PAUF-MycHis channel, imaged by STED) to VAP-A–OSBP–mediated ER–Golgi MCSs (BiFC channel, imaged by confocal microscopy). Images were acquired and deconvolved as described in Materials and methods. High magnifications of the boxed areas are shown in the right panels. Arrowheads indicate putative nascent CARTS located in the close vicinity of the BiFC signal of Vn-OSBP–Vc-VAP-A. Scale bars, 5 µm (left panels), 1 µm (right panels). (B) PAUF-MycHis secretion in control (Cont) and SCAP knockdown cells. The graph shows quantification of secreted PAUF-MycHis relative to the total cellular level and normalized as the values in control cells. Data are means ± SEM (n = 3 independent experiments; **, P < 0.01; unpaired two-tailed Student’s t test). IB, immunoblotted. (C) Biogenesis of mKate2-FM4-PAUF–containing CARTS in control and SCAP knockdown cells. The graph shows the number of mKate2-FM4-PAUF–containing CARTS in control and SCAP knockdown cells at 15 min after the temperature shift to 37°C. Data are means ± SEM (n = 20–29 cells per condition; ****, P < 0.0001; unpaired two-tailed Student’s t test). Scale bar, 10 µm. See also Video 1.

SCAP is required for the biogenesis of CARTS at the TGN. (A) Close proximity of CARTS formation sites (PAUF-MycHis channel, imaged by STED) to VAP-A–OSBP–mediated ER–Golgi MCSs (BiFC channel, imaged by confocal microscopy). Images were acquired and deconvolved as described in Materials and methods. High magnifications of the boxed areas are shown in the right panels. Arrowheads indicate putative nascent CARTS located in the close vicinity of the BiFC signal of Vn-OSBP–Vc-VAP-A. Scale bars, 5 µm (left panels), 1 µm (right panels). (B) PAUF-MycHis secretion in control (Cont) and SCAP knockdown cells. The graph shows quantification of secreted PAUF-MycHis relative to the total cellular level and normalized as the values in control cells. Data are means ± SEM (n = 3 independent experiments; **, P < 0.01; unpaired two-tailed Student’s t test). IB, immunoblotted. (C) Biogenesis of mKate2-FM4-PAUF–containing CARTS in control and SCAP knockdown cells. The graph shows the number of mKate2-FM4-PAUF–containing CARTS in control and SCAP knockdown cells at 15 min after the temperature shift to 37°C. Data are means ± SEM (n = 20–29 cells per condition; ****, P < 0.0001; unpaired two-tailed Student’s t test). Scale bar, 10 µm. See also Video 1.

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