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. 2012 Mar 12;7(3):e33297. doi: 10.1371/journal.pone.0033297

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Becker et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Panel A: Bone marrow-derived macrophages (BmM) were derived from bone marrow precursor cells of C57BL/6 mice cultured with M-CSF. Classically activated macrophages (M1) and alternatively activated macrophages (M2) were derived from BmMs by treatment with IFN-γ and LPS or with IL-4. Panel B: qRT-PCR of markers used to detect M1 and M2 macrophages. Results (means and SEMs, N = 6) were standardized to 18S, expressed relative to the cell type with the highest expression of each gene, and are representative of 3 independent analyses. Panel C: LC-ESI-MS/MS analysis of plasma membrane proteins isolated from differentially activated macrophages. Proteins were quantified by spectral counting (total number of peptides identified for a given protein) and subjected to sequential criteria to identify 192 plasma membrane proteins that were reproducibly detected with high confidence. Panel D: Quantification of the membrane proteomes of M1 and M2 macrophages. Differentially expressed proteins (red, upregulated; green, downregulated; gray, not significantly different) were identified based on t-test and G-test statistics. Significance cutoffs (dashed lines; p<0.05 and G-statistic >1.5 or <−1.5) were determined based on permutation analysis (estimated FDR<5%). Panel E: Quantification of the membrane proteomes of M1 macrophages and BmMs. Proteins differentially expressed by M1 cells relative to both BmMs and M2 cells are indicated with colored dots (red, upregulated; green, downregulated). Proteins differentially expressed by M1 and M2 cells (Panel D) but not differentially expressed by M1 and BmMs are indicated by gray dots. Panel F: Examples of proteins that distinguish M1 cells from both BmM and M2 cells (CSF1R, ITGAL). Results (N = 6 per group) are means and SDs. Panel G: Examples of proteins that fail to distinguish M1 cells from both BmM and M2 cells (CD14, ITGAV). Panel H: Plasma membrane proteins differentially expressed by M1 cells (36 proteins), M2 cells (35 proteins), and BmMs (17 proteins).