Figure 2. Proteomic analysis of protein abundance in WT vs.Alkbh7^-/-.

Hearts from young male WT or Alkbh7^-/- mice were analyzed by tandem mass tag LC-MS/MS as per the methods. (A) Volcano plot showing relative levels of 3737 proteins. X-axis shows Log₁₀ of fold change (Alkbh7^-/- / WT) and Y-axis shows Log₁₀ of significance (paired t-test, N = 5). Proteins crossing thresholds (gray lines) in upper left or right quadrants are labeled. (B) Activity of GLO-1 in WT or Alkbh7^-/- heart cytosol. (C) Activity of GLO-2 in WT or Alkbh7^-/- heart cytosol. (D) Western blot showing abundance of GLO-1 in WT or Alkbh7^-/- heart cytosol, with Ponceau stained membrane below. (E) Quantitation of GLO-1 blot, normalized to protein loading. Bar graphs in panels B/C/E show means ± SE, N = 4–5, with p-values (paired t-test) shown above error bars. (F) Schematic showing the methylglyoxal detoxification system and its relationship to glycolysis. Abbreviations: AGEs: Advanced glycation end products, GSH: glutathione. MGO: methylglyoxal. In bar graphs, N for each group is shown in parentheses.

Figure 2—figure supplement 1. Representative gel from FLAG-tagged ALKBH7 pull-down experiment.

Lane one shows control (empty vector) cells. Remaining four lanes are ALKBH7-FLAG cells treated with MMS for indicated times. Proteins were cut from lane 3 (1 hr. post-MMS) for identification (see Supplementary file 1 Table S1).

Figure 2—figure supplement 2. Enzyme activities in WT and Alkbh7^-/-tissues.

Activities of (top to bottom) citrate synthase, respiratory complex I, respiratory complex II, and α-ketoglutarate dehydrogenase, were measured spectrophotometrically as per the methods, in (A) Heart mitochondria, (B) Liver mitochondria. Panel (C) shows GLO-1 and GLO-2 activity in liver cytosol, complementing the heart data in Figure 2B/C. All data are means ± SEM, N = 4–5, p values (paired t-tests) are shown above the error bars. N for each experiment shown in parentheses.