2-D–DIGE analysis of TnI phosphorylation. Cardiac samples of Sham and AOB were analyzed by 2-D–DIGE. The first dimension was performed by electro focusing on IPTG strips pH range 7–11. The next dimension was performed on SDS-PAGE gels for molecular weight separation. A shows a representative 2-D–DIGE gel (pH 7–11) wherein Sham and AOB samples were tagged with Cy3 and Cy5 dyes. The TnI spots are indicated by the square box in the bottom right area of the 2-D gel. B illustrates strategies employed to identify the TnI gel spots of this sample. Top: Pan-total TnI antibody and Ser23/24 phospho-specific antibody Western blots. Middle: 2-D–DIGE analysis of untreated pooled samples stained with Cy5 and PP1, and PP2A-treated pooled samples stained with Cy3. Bottom: Sypro Ruby total protein and ProQ Diamond gel stains that identify phosphorylated proteins. From these results, we were able to identify the TnI gel spots as unphosphorylated (U), single (P₁), and double phosphorylated (P₂) as indicated. C shows an example of the 2-D–DIGE results where Sham and AOB differentially labeled samples are run together and analyzed by the GE Decyder 2-D–DIGE analysis package. The top panel illustrates the differential analysis based on simultaneous scanning of the Cy3 and Cy5 channels. The bottom bar graphs show the average (n = 7 for Sham and n = 7 AOB) level of unphosphorylated (U spot) and total phosphorylated (P₁ + P₂) cardiac TnI. On average, the phosphorylation level of TnI was slightly, albeit not significantly, decreased in the AOB group. Sham control, open bars; AOB, closed bars. Sham control vs. AOB: (+) TnI-P P = 0.299; (−) TnI-P P = 0.668). Source data are available for this figure: SourceData F3