Xenbase Image ID: 144186

Figure 2. Brefeldin A causes resorption of cis-Golgi structure to ER, but does not abolish the cilia targeting of P/rds. A, Western blot analysis of BFA (0.5 μg/ml for 4 h)-treated hTERT-RPE1 cells stably expressing bP/rds-HA-FLAG. Total cell lysate (T), and cell surface fraction (PM) isolated by biotinylation method were treated with Endo H (En). BFA treatment led to abolishment of the small amount of the Endo H-resistant form of P/rds on the cell surface. The arrowhead indicates the position of Endo H-processed P/rds. B, bP/rds (green) was colabeled with anti-GM130 (cis-Golgi, red) in the absence (Con) and presence (BFA) of BFA treatment. BFA treatment caused GM130 redistribution, which indicates the disruption of cis-Golgi structures. C, bP/rds (green) was colabeled with anti-acetylated tubulin (red) in the absence (Con) and presence (BFA) of BFA treatment. Cilia localization of bP/rds was maintained after BFA treatment. D, E, Trafficking of bP/rds-Dend2 or SSTR3, stably expressed in hTERT-RPE1 cells, was tested by FRAP in the absence (Con) and presence of (BFA) treatment. D, One hour after photobleaching, P/rds recovered in the cilia of control cells. P/rds recovered in 50% of cilia treated with BFA. Under the same conditions, SSTR3 recovery was blocked by BFA treatment. E, The amounts of cilia localized P/rds and SSTR3 immediately following and 1 h after photobleaching. The amount was normalized to the prebleach levels. Significant recovery of bP/rds-Dend2, but no significant recovery of SSTR3, was observed in BFA-treated cells. ***p < 0.001 by MWRST. The data are represented as mean ± SD. The numbers of cilia analyzed from at least three sample preparations are indicated in parentheses. F, Cells stably expressing bP/rds-Dend2 were treated with 150 μM cycloheximide for up to 24 h and then analyzed for the bP/rds-Dend2 protein level by Western blots. The half-life of bP/rds-Dend2 is estimated to be ∼1.8 h. The images are confocal images of a single x–y plane. Scale bars: B and C, 10 μm; D, 5 μm. Image published in: Tian G et al. (2014) Copyright © 2014. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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