Identified Proteins in GCH1 pull-down rat liver samples.

<p>Identified Proteins in GCH1 pull-down rat liver samples.</p

The interaction of GCH1 with GFRP in different organs.

<p>GCH1 and its interacting proteins were purified from brain, heart, liver and kidney, and analyzed by western blot against GCH1 antibody (A) and GFRP antibody (B). EL1 is the first eluate, EL2 is the second eluate. Eluates from IgG conjugated column were used as controls. Protein lysates from different organs were immunoblotted with GFRP antibody and HSP90. HSP90 was used as a loading control (C). HEK cells stably over-expressing GCH1 (GCH1-HEK) were immunoprecipitated with IgG or GCH1 antibody and immunoblotted against GFRP and GCH1. Straight cell lysate of GCH1-HEK cells (Lysate) was also loaded for comparison (the first lane). Liver homogenate was used as a positive control (D). HEK cells were transiently transfected with Flag-GCH1, HA-GFRP or pcDNA, and immunoprecipitated with Flag tag (E) or HA tag and immunoblotted with GCH1 and GFRP antibodies. In (E), cell lysates (the first lane) from HEK cells transfected with FLAG-GCH1 and HA-GFRP were used as positive controls for GFRP and GCH1 expression.</p

Purification of GCH1 and its interacting proteins.

<p>(A) Flow chart showing the procedure for purification and characterization of GCH1 complexes from rat organ (brain, heart, liver and kidney). (B) Protein samples from different steps of protein purification by IgG and GCH1 from rat liver were separated by SDS-PAGE, (B) stained with Gelcode Blue and (C) analyzed by Western blot analysis against GCH1 antibody. IgG and GCH1 purified samples from rat heart were (D) silver stained and (E) analyzed by Western blot analysis against GCH1. MK, molecular weight marker; Ig, IgG; GCH, GCH1; FL, flow-through; FLG, Flag tag, Flg-GCH is cell lysates from FLAG-GCH1 over-expressing HEK cells, used as positive control.</p

The interactions of GCH1 with its 6 protein partners identified by ESI/LC/MS were validated in rat liver by western blot analysis.

<p>The purified GCH1 complexes from rat liver were separated by SDS-PAGE and then immunoblotted with different antibodies as indicated. The blots were representatives of five independent biological repeats.</p

The interaction of rat liver GCH1-interacting proteins with GCH1 in brain, heart and kidney determined by western blot analysis.

<p>(A) The protein lysates from different rat organs were immunoblotted with antibodies indicated. (B) The GCH1 protein complexes purified from different organs were immunoblotted with the indicated antibodies.</p

RT-PCR results for NOX1.

<p>Both control and NEC samples increased from D0 to D1. There were no same-day differences between control and NEC. n = 4–5 per group. *  =  p<0.05.</p

Immunofluorescence for p47^phox and GP91^phox on D1, D2, and D4.

<p>Fig. 8a shows control samples, and Fig. 8b shows NEC samples. The images are of p47^phox (red) GP91^phox (green), and the co-localization of the two proteins (yellow).</p

RT-PCR results for eNOS.

<p>There were no same-day differences between control and NEC. n = 4–6 per group. *  =  p<0.05.</p

NADPH-dependent O₂^•– production in small intestinal homogenates from D4.

<p>n = 5–7 per group F =  Formula, F/H  =  Formula and hypoxia, F/L  =  Formula and LPS, F/H/L  =  Formula, Hypoxia, and LPS (NEC model). *  =  p<0.05 between NEC and each group.</p

Effects of inhibitors on NADPH- dependent O₂^•– production on D4 small intestinal homogenates.

<p>n = 6 for control, 5 for NEC. *  =  p<0.05</p