The dynamic of membrane association and dissociation of Sec31 is reduced with the non-phosphorylatable mutant.

<p>(A) Shown are the results of fluorescence recovery after photobleaching (FRAP) of Sec31 and its 4SA<b>.</b> GFP-tagged wild type Sec31 (WT) and the 4SA mutant were expressed transiently in HeLa cells. A GFP positive dot was photobleached and subsequent fluorescence recovery was monitored for 30 s with 1.2 s intervals. Eight dots were photobleached per cell and results were normalized. Represented are the average of ∼7 experiments. Bars, SEM. (B) The representative images at the indicated time points of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054382#pone-0054382-g003" target="_blank">Figure 3A</a>. Sizes, 1.85 (w) x 1.85 (h) µm.</p

Membrane associated Sec31 is not phosphorylated.

<p>HeLa cells were subjected to subcellular fractionations to prepare cytosol (Sup) and membranes (Ppt) by ultracentrifugation. The recovery of the transmembrane protein, calnexin in the membrane fraction (Ppt) indicates that the fractionation was performed properly. Subsequently, Sec31 from both fractions were immunoprecipitated and subjected to western blotting with anti-Sec31 and anti-phospho-serine/threonine antibodies.</p

Kinetics of Sec31 turnover at single ERES.

*<p>Calculated using the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054382#pone.0054382.e001" target="_blank">equation 1</a> in the text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054382#pone.0054382-Forster1" target="_blank">[48]</a>.</p>**<p>Calculated using the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054382#pone.0054382.e002" target="_blank">equation 2</a>, which is the reaction dominant model described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054382#pone.0054382-Sprague1" target="_blank">[49]</a>.</p

Unraveling new roles for serotonin receptor 2B in development: key findings from Xenopus

The serotonin receptor 5-HT2B has been shown to be critically important during embryogenesis as the knockout of this gene in mice causes heart defects and embryonic lethality that impairs further analyses on other embryonic cell and tissue types. In the present review we highlight how the use of Xenopus laevis, an alternative vertebrate model suitable for gene loss and gain of function analyses, contributed to our understanding of the role of 5-HT2B signaling during development. In vivo studies showed that the 5-HT2B signaling is not only required for heart development but also has a crucial role in ocular and craniofacial morphogenesis being involved in shaping the first branchial arch and the jaw joint, in the retinogenesis and possibly in the periocular mesenchyme development. These findings may be relevant for our understanding of congenital defects including human birth malformations. In addition, the 5-HT2B appears to be required for the therapeutic actions of selective serotonin reuptake inhibitors commonly prescribed as antidepressant drugs to pregnant and lactating women. We discuss how the understanding of the molecular basis of the serotonin signaling in a suitable animal model embryogenesis may open new lines of investigations and therapies in humans

Additional file 1: Figure S1. of ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic

Aggregation of ERES fluorescence puncta during active autophagy. Boxed areas of the pericentriolar ERES from cells grown in growth medium (left) and amino acid starved medium (right) were plotted as a function of the XY position of each pixel from the boxed area. The number of signal peaks was reduced in amino acid starved medium but the size of the peaks was larger, indicating aggregation of ERES signals into fewer and brighter fluorescence puncta. (TIFF 4537 kb

Giantin RNAi did not change the cisternal lengths of Golgi mini-stacks generated by nocodazole treatment.

<p>(A) Nocodazole, giantin siRNA-, and mock-treated cells were processed for electron microscopy. Bar, 333 nm. (B) Average length of Golgi cisternae in a Golgi stack in approximately 10 different cells. Bars represent SEM (n = ∼10 cells).</p

Additional file 6: Figure S6. of ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic

The interactions between the ULK1 phosphorylation mutants of Sec23A and Sec31A. (A) S207 and T405 mutants of Sec23A were tested for their interaction with Sec31A by co-expressing the Myc-His-Sec23A mutants and GFP-Sec31A. (B) Combinations of S207 and T405 double mutants were tested for the interaction with Sec31A. Overexpression of the indicated protein was carried in HEK293T cells, followed by immunoprecipitation of Myc-His-Sec23A by anti-Myc antibody. Co-precipitated GFP-Sec31A was detected by anti-GFP antibody. (B) Wildtype Sec23A, S207A and T405A mutants were tested for the interaction with GFP-Sec31A in growth medium or in amino acid starved medium EBSS. (TIFF 5305 kb

Giantin RNAi enhanced anterograde trafficking and changed surface glycosylation patterns.

<p>(A) Giantin siRNA- and mock-treated cells were transfected with VSV-G-tsO45-YFP and incubated at the restrictive temperature of 40°C overnight before shifting them to the permissive temperature of 32°C. Cells were surface labeled for VSV-G. Cell images were captured and analyzed using Image J and Photoshop (n = ∼20 cells). Error bars represent SEM of three independent experiments. (B) SEAP was increased by giantin RNAi. HeLa cells stably expressing SEAP were transfected with siRNAs. After 90 h, the cells were washed and fresh culture media were added. After 6 and 24 h, respectively, culture supernatants were collected and processed for SEAP activity measurement. The ratio of the activities after 6 and 24 h are shown in the graph. Bars, SD (n = 3). (C) Giantin siRNA- or mock-treated cells were surface-labeled with FITC-lectins and analyzed by flow cytometry. SucWGA does not bind to sialyl-sugar moieties, unlike the native form, but retains its specificity to N-acetylglucosamine.</p

AMPK inhibitor, Compound C, abolishes the lymphocyte-facilitation of tight junction assembly.

<p>(a) Representative pictures of ZO-1 relocation to cell–cell junctions in MDCK cells cultured alone and co-cultured with lymphocytes, with or without the AMPK inhibitor, Compound C, (A. MDCK; B. Co-culture; C. Co-culture+Compound C; D. Co-culture+DMSO) at various time points (0h, 0.5h, 1h, 2h) after calcium switch. (Scale bar: 30 µm). (b) Quantification of ZO-1 relocation to cell–cell junctions (length of ZO-1/cell (µm)) in MDCK cells cultured alone and co-cultured with lymphocytes, with or without AMPK inhibitor, Compound C, at various time points (0h, 0.5h, 1h, 2h) after calcium switch. The asterisks denote significant differences detected in (C) Co-culture+Compound C vs. (D) Co-culture+DMSO by Student's <i>t</i> test (*, p<0.05; **, p<0.01).</p

AMPK is activated in the epithelium-lymphocyte co-culture and by TNF-α.

<p>(a) Western blot results showing increased level of AMPK phosphorylation in MDCK-lymphocyte co-culture and Calu-3-lymphocyte co-culture as compared to MDCK and Calu-3 cultured alone, respectively. Total AMPK remained unchanged, indicating that AMPK is activated in these co-cultures, with beta-actin used as loading control. The corresponding statistical analysis is on the right panel (*, p<0.05; ***, p<0.001). (b) ATP assay showing no significant changes of cellular ATP levels in MDCK-lymphocyte co-culture vs. MDCK cells alone (‘ns’ stands for no significance). Cell lysates from MDCK cells with and without lymphocytes were subjected to an ATP assay. The ATP contents were normalized to the total protein amount. Data were from three experiments. (c) Lysates prepared from MDCK cells maintained in α-MEM in the absence or presence of TNF-α were blotted with the indicated antibodies. Treatment with AICAR served as the positive control for AMPK activation. (d) The activated AMPK was represented as a relative ratio to total AMPK. The phosphorylation of AMPK is significantly increased by exposure for 2 hours to TNF-α treated, as assessed by <i>Student's test</i> (**, p<0.01).</p