GTPCH1 non-covalently interacts with polyubiquitin in mouse heart and lung <i>in vivo</i>.

<p>Mice tissues (n = 4) were isolated and the homogenates were generated as described in Experimental Procedures. The tissue lysates were incubated with UIM-agarose for ubiquitin affinity precipitation (AP). The proteins that interacted with polyubiquitin chains were analyzed by western blot using specific antibodies. <b>A.</b> GTPCH1 and polyubiquitin chains interaction in mouse heart and <b>B.</b> mouse lung. Control beads without UIM were used as a negative control are included in all experiments. The black triangle (▸) indicates the bound GTPCH1 by polyubiquitin chains and the asterisk (*) indicates a non-specific band. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043306#s3" target="_blank">Results</a> were obtained from four mice.</p

Inhibition of the 26S proteasome causes the accumulation of the GTPCH1/Ub complex.

<p><b>A and B.</b> HEK293 cells were transfected with FLAG-GCH1 or empty vector (control) for 2 days and treated with the 26S proteasome inhibitors MG132 or lactacystin (Lac, irreversible inhibitor) for 6 hours. Lysates from cells expressing FLAG-GTPCH1 were immunoprecipitated with anti-FLAG resin and immunoblotted using the indicated antibodies. HEK293 transfected with empty vectors were used as a negative control. The asterisk (*) indicates IgG bands. <b>C.</b> HEK293 cells were co-transfected with FLAG-GCH1 and HA-Ub plasmids and treated with or without MG132 for 6 hours. Lysates from cells expressing FLAG-GTPCH1 were immunoprecipitated with anti-FLAG resin and immunoblotted with the FLAG, HA, or β-actin antibodies. The blot shown is representative of three independent experiments.</p

Interaction of GTPCH1 with Lys48 linked polyubiquitin chains promotes its degradation.

<p><b>A.</b> HEK293 cells were co-transfected with FLAG-GCH1 and HA tagged ubiquitin plasmids (HA-Ub) for 2 days, cells were lysed, and the proteins were analyzed by immunoblotting using the indicated antibodies. The double plus symbol (<b>++</b>) indicates two folds of HA-Ub plasmids. HEK293 cells were co-transfected with FLAG-GCH1 plasmids and <b>B.</b> Lys48-only ubiquitin plasmids (K48 only-Ub) or, <b>C.</b> Lys63-only ubiquitin plasmids (K63 only-Ub). <b>D.</b> Compare of the GTPCH1 level with or without ubiquitin plasmids overexpression. The blot shown is representative of three independent experiments. Data are shown as mean±standard deviation (n = 3). *^#<i>P</i><0.05 vs. control (without ubiquitin plasmids overexpression). NS indicates <i>P</i>>0.05 vs. control.</p

GTPCH1 binds polyubiquitin in cultured cells.

<p><b>A.</b> Endogenous GTPCH1 binds polyubiquitin in mouse endothelial cells (SVEC4-10). Mouse endothelial cells were grown to confluence and cell lysates were incubated with UIM-agarose for ubiquitin affinity precipitation (AP). The interaction of GTPCH1 with polyubiquitin chains was analyzed by immunoblotting. The black triangle (▸) indicates the bound GTPCH1 by polyubiquitin chains. Control beads without UIM were used as a negative control. <b>B.</b> Co-immunoprecipitation of GTPCH1 with polyubiquitin in GTPCH1 overexpressed HEK293 cells. FLAG-GCH1 plasmids or empty vectors were transfected into HEK293 cells for 2 days. Cell lysates were incubated with the ubiquitin antibody and immunoprecipitated using protein A/G beads. The asterisk (*) indicates IgG bands. HEK293 transfected with empty vectors were used as a negative control. <b>C.</b> Polyubiquitin chains were co-immunoprecipitated with GTPCH1. HEK293 cell lysates expressing FLAG-GTPCH1 or control vector were incubated with anti-FLAG resin. Proteins that co-immunoprecipitated with FLAG-GTPCH1 were analyzed by immunoblotting using the indicated antibodies. The blot shown is representative of five independent experiments.</p

The ubiquitin-binding domain (UBD) of GTPCH1 mediates the interaction with ubiquitin chains.

<p><b>A</b>. Alignment of ubiquitin binding domains found in multiple proteins. The accession numbers are for GenBank (gi) and residue numbers of the sequences are shown on the left. Below, (*) indicates conserved hydrophobic core residues and (#) indicates conserved “GFP-loop”. Abbreviations used: HS, human; SC, yeast; MM, mouse. <b>B</b>. Schematic of the GTPCH1 deletion mutants. <b>C & D</b>. GST-tagged GTPCH1 (42–250) or deletion mutants (NΔ1∼NΔ3) were expressed in <i>E. coli</i>. Purified GST-GTPCH1 proteins bound to GSH beads were incubated with the synthesized tetraubiquitin chains (Ub4). The bound proteins were detected by immunoblot using the ubiquitin antibody. GST alone was used as a negative control. The blot shown is representative of three independent experiments. Data are shown as mean±standard deviation (n = 3). *<i>P</i><0.05 vs. control. NS indicates <i>P</i>>0.05 vs. control.</p

WeatherCases.m

A function m-file named WeatherCases.m related to comparison of two cases (a region with multiple SWMPs

Para_settings.m

M-file named Para_settings.m related to sensitive analysi

WNV_SWMP_R0.mv

A maple file named WNV_SWMP_R0.mw to show the variation of basic reproduction number R_0 along with combinations of weather conditions or SWMP properties

Weather.m

A function m-file named Weather.m to define a weather-driven dynamical syste

R0.m

A function m-file named R0.m related to sensitive analysi