Integrated Functional Analysis of the Nuclear Proteome of Classically and Alternatively Activated Macrophages

Figure 2

Schematic of workflow for developing the integrated proteome of M1- and M2-polarized macrophages. Mouse bone marrow Mφ were incubated for 18 h with IFN-γ/LPS and IL-4 to generate M1- and M2-polarized Mφ, respectively. Nuclear fractions were isolated as described in Materials and Methods. Each sample ( per group) was divided into two aliquots. One aliquot was treated with ascorbate (Asc⁺) to reduce SNO cysteines, and in the second aliquot, SNO was stabilized with neocuproine but not treated with ascorbate (Asc⁻). All aliquots were incubated with BODIPY FL N-(2-aminoethyl) maleimide (labels reduced cysteine) and resolved by two-dimensional electrophoresis (2DE). After imaging the gels for BD staining and BD spillover, each gel was stained with Pro-Q Diamond (detects phosphorylated proteins) and imaged. The differential protein abundance, SNO modification, and phosphorylation of protein spots were determined by ratiometric calculations. The fold changes in protein spots in all gels were subjected to statistical analysis, and protein spots that changed in any one of the parameter by -fold at were submitted to mass spectrometry analysis for protein identification. The proteome datasets were submitted to Ingenuity Pathway Analysis for network inquiry.