Fig 1.
Experimental workflow design, and proteomics data acquisition and analysis.
(A). Experimental workflow design. (B). Proteomics data acquisition and downstream analysis.
Table 1.
The effect of insulin on Akt2 interaction partner ROCK2.
Peak area (PA) for each protein is normalized against Akt2 PA in the same sample.
Fig 2.
Significantly enriched canonical pathways for the 49 significant Akt2 interaction partners identified in this study.
Pathway analysis was revealed by proteomics data and Ingenuity Pathways Analysis (IPA). The number of identified Akt2 interaction partners for a given pathway in this study are denoted besides each bar.
Fig 3.
Significantly enriched biofunctions for the 49 Akt2 protein interaction partners in L6 myoblasts.
The number of proteins identified for each biofunction is indicated in bold font, and the number of proteins for each biofunction with increased binding to Akt2 following the insulin treatment is indicated in the inset circle of each biofuntion, along with the –log(p-value) denoted inset the oval.
Table 2.
Significantly changed Akt2 protein interaction partners in L6 myoblasts upon insulin stimulation (n = 4, P<0.05).
Each protein was detected in > 4 of the samples as well as not detected in any of the NIgG samples.
Fig 4.
Insulin-stimulated association between Akt2 and ROCK2 in L6 myoblasts.
L6 myoblasts were serum-starved for 4h and stimulated with or without insulin (100 nM) for 15 min at 37°C. The cells were lysed and 1 mg of lysates were immunoprecipitated (IP) with normal IgG (NIgG) or Akt2 antibody and western blotted with anti-Akt2 and anti-ROCK2, (A); and relative abundance detected by HPLC-ESI-MS/MS (B).
Fig 5.
Significantly enriched canonical pathway, EIF2, for the Akt2 interaction partners identified in this study.
Pathway analysis was revealed by proteomics and Ingenuity Pathways Analysis. Akt2 is highlighted in purple. Proteins with increased interaction to Akt2 after the insulin treatment are highlighted in green, proteins with decreased interaction to Akt2 after the insulin treatment are highlighted in red, and identified interaction partners with no differences in their interaction to Akt2 under the basal and insulin stimulated conditions are highlighted in yellow. Proteins without color are identified in the network IPA database, but were not identified in this study.