Schematic diagram of a two-step crosslinking approach for identification of Akt binding partners.

<p>Akt antibody was immobilized on protein A/G beads via DSS (succinimidyl suberate) cross-linking. Following the incubation of the cell lysate with the immobilized Akt antibody, DSP (dithiobis[succinimidylpropionate]), a cleavable crosslinker, was used to produce stable complexes between Akt and its binding partners. The co-immunoprecipitated complexes were eluted with Laemmli buffer containing 5% β-mercaptoethanol. The liberated proteins were subjected to SDS-PAGE, tryptic digestion, and MS-based analysis followed by western blot validation.</p

Akt binding partners identified from Neuro 2A cells using a two-step crosslinking-co-IP/MS approach.

#<p>None of these proteins were detected without using DSP-crosslinking, with the exception of ERK1/2. Without DSP-crosslinking ERK1/2 was identified by only two distinct peptides (G<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061430#pone.0061430-TrinkleMulcahy1" target="_blank">[14]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061430#pone.0061430-Harlow1" target="_blank">[22]</a>R and A[193–201]K) with 5% sequence coverage.</p>a<p>Proteins found to be more associated with active Akt than inactive Akt by western blot analysis.</p>b<p>Association with Akt found to be independent of the activation status of Akt revealed by western blot analysis.</p>c<p>peptides identified by Bioworks (3.1).</p>d<p>XC score.</p

Identification of Nudc and TFII-I as potential Akt binding partners.

<p>Representative MS/MS spectra of Nudc (a) and TFII-I (b) peptides identified from the co-IP products. The sequence of peptides was assigned with a single letter abbreviation based on the fragment ions observed for the peptide segments. N-terminal b ions and C-terminal y ions resulting from the amide bond cleavage are labeled. * denotes the addition of 145.0198 Dalton due DSP-crosslinking. Western blotting indicated that Nudc co-immunoprecipitated with Akt in Neuro 2A cells (a). nc, negative control using IgG instead of Akt antibody at a similar protein level.</p

IGF-independent interaction of Akt with ERK1/2 or Nudc revealed by western blot analysis.

<p>The active Akt status was indicated by phosphorylation of T308. Con, control sample without IGF stimulation. IGF-stimulated samples were obtained by treating Neuro 2A cells with IGF at 10 ng/mL for 10 min. nc, negative control using IgG instead of Akt antibody at a similar protein level.</p

Representative MS/MS spectra of Akt and ERK1/2 peptides obtained from the co-IP complex.

<p>MS/MS analysis of the doubly charged ion at m/z 912.46 or 798.90 originated from the peptide segment Y<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061430#pone.0061430-Amoresano1" target="_blank">[26]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061430#pone.0061430-Novina1" target="_blank">[39]</a>K of Akt (a), or A[193–205]K of ERK1/2 (b), respectively. The sequence of the peptide was assigned with a single letter abbreviation based on the fragment ions observed for the peptide segments. N-terminal b ions and C-terminal y ions resulting from the amide bond cleavage are labeled. * denotes the addition of 145.0198 Dalton due DSP-crosslinking.</p

Western blot analysis of Akt and ERK1/2 monitored during co-IP procedures.

<p>a. Chemical structures of the crosslinkers used in the study. b. Antibody contamination was eliminated by DSS-crosslinking of Akt antibody to protein A/G agarose beads. c. ERK1/2 co-immunoprecipitation with Akt was significantly improved by DSP-crosslinking. d. DSS-crosslinking did not alter antibody-antigen binding. nc, negative control using IgG instead of Akt antibody at a similar protein level.</p

IGF-dependent interaction of Akt with EBP1.

<p>a, Western blot analysis indicating that active Akt is associated with EBP1 more than inactive Akt. The active Akt status was indicated by phosphorylation of T308. b, Western blot analysis of the co-IP complex with or without DSP-crosslinking following the incubation of the IGF-stimulated cell lysate with Akt antibody immobilized on the beads by DSS. EBP1 was negligibly detected in the IP product without the use of DSP-crosslinking. c, Representative MS/MS spectrum of a peptide corresponding to EBP1 identified only with DSP-crosslinking approach. The sequence of the peptide was assigned with a single letter abbreviation based on the fragment ions observed for the peptide segments. N-terminal b ions and C-terminal y ions resulting from the amide bond cleavage are labeled. Con, control sample without IGF stimulation. IGF, IGF-stimulated samples obtained by treating Neuro 2A cells with IGF at 10 ng/mL for 10 min. nc, negative control using IgG instead of Akt antibody at a similar protein level. * denotes the addition of 145.0198 Dalton due DSP-crosslinking.</p

Omega-3 fatty acid deficiency impairs recovery from TBI-induced motor deficits.

<p>(A) The rotarod test showing slower recovery from TBI in the DHA deficient group (Deficient TBI) as compared to the adequate (Adequate TBI) mice with statistically significant differences on day 2 and day 4 after TBI (* <i>p</i><0.05 and *** <i>p</i><0.001 vs. the respective O-3 adequate group; <i>n = </i>8). (B) The beam walk test showing greater hindlimb footslips in DHA deficient mice as compared to the respective adequate controls (* <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 vs. adequate TBI group; <i>n = </i>7–8).</p

Composition of omega-3 fatty acid adequate and omega-3 fatty acid deficient diets¹.

<p>1 These diets were prepared by Dyets Inc. (Bethlehem, PA, USA) based on AIN-93G (18) and have been used to achieve DHA depletion in rodents (24,36).</p><p>2 DHASCO: DHA Algal Oil.</p><p>3 tBHQ: tert-butylhydroquinone.</p

Depletion of DHA does not affect the volume of the lesion induced by TBI.

<p>Lesion volumes of DHA depleted brains after TBI are not statistically different from those of the DHA adequate controls (<i>n</i> = 6).</p