UNC5B expression in murine tissue.

<p><b>A)</b> Relative protein expression in murine brain (Br), intestine (In), liver (Li), heart (He) and kidney (Ki) by Western Blot analysis (n = 3; pooled samples). <b>B)</b> Gating of leukocytes subpopulations such as lymphocytes, monocytes and neutrophil granulocytes. <b>C)</b> Percentage of positive gated neutrophil granulocytes, leukocytes, monocytes and T- and B-cells (n = 5). Representative histograms of UNC5B expression (red) of CD45+ leukocytes and CD15+ neutrophils are shown <b>D)</b> Immunofluorescence UNC5B (green = Alexa 488) co-staining on neutrophil leukocytes (CD45 marked) and granulocytes (CD15 marked). Merged images show the localization of UNC5B on the neutrophils and leukocytes (n = 3 per group).</p

SiRNA in-vivo knockdown of UNC5B attenuates myocardial IR injury in WT mice.

<p><b>A)</b> Infarct size of mice with UNC5B specific siRNA treatment compared to mice received non-targeting siRNA after 60 minutes of myocardial ischemia followed by 2 hours reperfusion. Calculated is the percentage of necrotic tissue to AAR. <b>B)</b> and <b>C)</b> Correlating serum Troponin I and IL-6 levels of these mice (n = 6 per group; *<i>P</i><0.05; ***<i>P</i><0.001 as indicated). <b>D)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted tissue) of the siRNA treated mice.</p

Functional inhibition of UNC5B reduces damage in myocardial IR.

<p><b>A</b>) Infarct size after IR in WT mice receiving antibody against UNC5B (2.5 µg/mouse) iv. 30 min before onset of surgery. Controls were treated with corresponding IgG antibody. Calculated is the percentage of necrotic tissue to AAR. <b>B)</b> and <b>C)</b> Correlating serum Troponin I and IL-6 levels of these mice. (n = 6 per group; *<i>P</i><0.05; ***<i>P</i><0.001 as indicated). <b>D)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted tissue) of the antibody treated mice.</p

Functional inhibition of UNC5B after depletion of neutrophil granulocytes does not results not in additional cardioprotection.

<p><b>A)</b> Infarct size following IR in WT and UNC5B^+/− mice after neutrophil granulocyte depletion and subsequent anti- UNC5B antibody adminsitration. Calculated is the percentage of necrotic tissue to AAR. <b>B)</b> and <b>C)</b> Correlating serum Troponin I and IL-6 levels of these mice. (n = 4 per group). <b>D)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted necrotic tissue.</p

Myocardial infarction is diminished in <i>UNC5B^+/−</i> mice.

<p><b>A)</b> Comparison of UNC5B protein expression in <i>UNC5B^+/−</i> and WT animals in different organs (n = 5 per group). <b>B)</b> Comparison of UNC5B transcriptional mRNA levels of organs normalized to brain tissue of WT animals (n = 5 per group). <b>C)</b> Infarct size in <i>UNC5B^+/−</i> mice compared with WT mice after 60 minutes of myocardial ischemia followed by 2 hours reperfusion. Calculated is the percentage of necrotic tissue to AAR. <b>D)</b> and <b>E)</b> Correlating serum troponin I and IL-6 levels of <i>UNC5B^+/−</i> and WT mice (n = 6 per group; *<i>P</i><0.05; **<i>P</i><0.01 as indicated) <b>F)</b> Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infarcted tissue).</p

Transendothelial neutrophil migration is inhibited by anti- UNC5B antibody.

<p><b>A</b>) PMN transmigration studies were performed in transwells using fMLP as chemoattractant in the lower compartment. PMNs were pre-incubated with either anti-UNC5B antibody or IgG control prior to transmigration. Number of transmigrated PMNs was assessed by MPO measurement. <b>B</b>) Passive flux of FITC Dextran across HMEC endothelium monolayers. Flux was detected by measurement of passed FITC Dextran in the basal compartment (n = 6 per condition; *<i>P</i><0.05 as indicated).</p

Co-immunoprecipitation and overlay assays indicate interaction of 14-3-3 and IRS-2 upon IGF-1/insulin stimulation.

<p><i>A.</i> HEK293 cells were transiently transfected with GFP or GFP-IRS2 and after serum starvation cells were incubated with 50 ng/ml IGF-1 for 30 min or after preincubation with 100 nM wortmannin for 30 min. 400 µg total protein was used for immunoprecipitation with 14-3-3 antibody (C-17) and samples were separated on 5–15% gradient gel. Upper membrane was incubated with IRS-2 antibody, lower membrane with 14-3-3 antibody (K-19). <i>B.</i> HEK293 cells were transfected with GFP-IRS2 and stimulation was carried out after starvation for serum overnight with 50 ng/ml IGF-1 for 30 min or subsequently after preincubation with 1 µM PI-103 for 30 min. 250 µg of total protein was pulled down using GFP-Trap®. SDS-PAGE followed transfer onto nitrocellulose membranes. Overlay assay followed stripping of the membrane and reprobing with GFP antibody as loading control. <i>C.</i> Extent of interaction was quantified by scanning densitometry of blots and normalization for GFP-IRS2 serum starved condition (mean ± SEM; n = 4; *p<0.05 serum starved vs. IGF-1 or IGF-1 vs. PI-103/IGF-1). <i>D</i>. Male C57Bl/6 mice were fasted overnight and injected intravenously with 2 IU (international units) insulin. After 10 min liver was taken and 500 µg of total protein was immunoprecipitated with IRS-2 antibody. After performing overlay assay membrane was stripped and reprobed with IRS-2 antibody as loading control. Two mice of each group are shown. <i>E</i>. Densitometric analyses of 14-3-3 interaction with IRS-2. Overlay signal was normalized for total IRS-2 protein content (mean ± SEM; n = 4; *p<0.05 fasted vs. insulin). <i>F</i>. Male C57Bl/6 mice were fasted overnight, refed for 4 hours or injected intraperitoneally with insulin for 30 min. Procedure as in <i>G</i>. Four mice of each group are shown. <i>G</i>. 14-3-3 interaction with IRS-2 was quantified by scanning densitometry of immunoblots and normalization for IRS-2 protein (mean ± SEM; n = 4; *p<0.05 fasted vs. refed and insulin stimulation).</p

The area spanning amino acids 301–600 on IRS-2 is the 14-3-3 binding region.

<p><i>A.</i> Schematic illustration of truncated IRS-2 constructs to identify the 14-3-3 binding region. <i>B.</i> 20 µg of protein was separated on a 5–15% gradient gel and membrane was probed with GFP antibody to check expression and molecular weight of truncated IRS-2 versions. The arrow indicates a longer exposure time. <i>C.</i> HEK293 cells were transfected with either GFP-IRS2 or truncated versions of IRS-2 (GFP-IRS2-1-300, GFP-IRS2-1-600, GFP-IRS2-301-1321, GFP-IRS2-601-1321, GFP-IRS2-301-600), starved for serum overnight and stimulated for 30 min with 50 ng/ml IGF-1 or subsequently after preincubation with 1 µM PI-103 for 30 min. With GFP-Trap® 250 µg protein was pulled down and samples were subjected to overlay assay. For loading and expression control membranes were stripped and reprobed for GFP.</p

Inhibition of Akt/PKB reduces IGF-1-induced phosphorylation of serine 573 and 14-3-3 binding.

<p><i>A</i>. Flp-In HEK293 cells stably expressing GFP-IRS2 were starved for serum and incubated for 30 min with 50 ng/ml IGF-1 or 1 µM Akti-1/2 alone, or Akti-1/2 preincubation followed IGF-1 stimulation. 100 µg of total protein was separated on 7.5% SDS gels and membranes were probed with p-Ser-573 and p-Thr-308 of Akt/PKB. Membranes were stripped and reprobed with respective antibodies for detection of protein levels. <i>B</i>. Effect of Akt/PKB inhibition on serine 573 phosphorylation was assessed by scanning densitometry of blots and normalization for protein (mean ± SEM; n = 3; *p<0.05 IGF-1 vs. Akti/IGF-1). <i>C</i>. GFP pulldown from Flp-In HEK293 cells stably expressing GFP-IRS2, stimulated as in <i>A</i>. 200 µg protein was pulled down and overlay assay followed reprobing with IRS-2 antibody. 100 µg of total protein was separated on 7.5% SDS gel and membrane was checked for p-Thr-308 phosphorylation and reprobed with Akt/PKB protein antibody.</p

Ser-573 influences phosphorylation of Akt/PKB.

<p><i>A</i>. HEK293 cells transiently expressing GFP-IRS2 or GFP-IRS2-S573A were stimulated with 50 ng/ml IGF-1 for the indicated time points. 40 µg of total protein was separated on 7.5% SDS gels and membranes were incubated with GFP antibody to ensure equal expression levels and with p-Thr-308 and p-Ser-473 antibody respectively. Corresponding Akt/PKB reblots are shown. <i>B</i>. Densitometric analyses of Akt/PKB phosphorylation. Black diamonds represent IRS-2 wild type, white squares IRS2-S573A mutant. Phosphorylation was normalized against total protein and IRS-2 wild type stimulated with IGF-1 for 5 min was set as 1 (mean ± SEM; n = 3; *p<0.05 IRS-2 120 min vs. S573A 120 min; IRS-2 240 min vs. S573A 240 min).</p