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. 2010 Dec 1;21(23):4130–4140. doi: 10.1091/mbc.E10-01-0022

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 1. — Arf1+Arf4 double knockdown induces ERGIC tubules. (A) The protein level of both Arf1 and Arf4 (lower arrowheads) was reduced to ∼20% in HeLa cells stably expressing GFP-ERGIC-53 and cotransfected with Arf1+Arf4 shRNAs. Lysates were blotted with anti-Arf1, anti-Arf4, or as a loading control with anti-tubulin. (B) Arf1+Arf4 knockdown induces GFP-ERGIC-53-positive tubules (arrows), which connect ERGIC clusters (bottom panel, filled arrowheads). Such connections are restricted to some ERGIC clusters in knockdown cells and are absent in control cells (bottom panel, empty arrowheads). Bottom panels are magnifications of the highlighted regions. (C) Time series of live cells imaged every ∼3 s (from Supplemental Movie S1) where stationary ERGIC clusters connected (filled arrowheads) or not (empty arrowheads) by tubules are observed. The bottom panel is a magnification. (D–G) Confocal sections of knockdown cells as described in B stained for CLIMP-63 (D), Sec31 (E), GPP130 (F), or α-tubulin (G). GFP-ERGIC-53 do not colocalize with CLIMP-63, Sec31, or GPP130 (empty arrowheads in D-F). In some instances GFP-ERGIC-53-positive tubules colocalize with Sec31 spots or α-tubulin tubules (filled arrowheads in E and G). Insets and bottom panels are a magnification of the highlighted regions. pSUPER and Arf1 shRNA vectors carry nucECFP. Transfected cells were identified by nucECFP signal. Bars, 5 μm.