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

Figure 8

Verification of changes in abundance and phosphorylation levels of two proteins in polarized Mφ. Macrophages were incubated for 18 h with IFN-γ/LPS or IL-4 to differentiate to proinflammatory (M1) and anti-inflammatory (M2) activation state, and nuclear fractions were prepared as described in Materials and Methods. (a) Proteome dataset for changes in abundance and phosphorylation of hnRNPA2/B1 and hnRNPA3. (b) Representative Western blot images are shown (3 biological replicates per group) for nuclear abundance of hnRNPA2/B1, hnRNPA3, and Lamin B (nuclear loading control) in M0, M1, and M2 Mφ. We used 5 μg, 6 μg, and 12 μg of each of the nuclear fractions for Western blotting for hnRNPA2/B1, hnRNPA3, and Lamin B, respectively. (c, d) Bar graphs show densitometry analyses for hnRNPA2/B1 and hnRNPA3 abundance in nuclear fractions of Mφ, normalized to Lamin B abundance (6 biological replicates per group in two experiment sets). (e) Nuclear fractions (500 μg each, three biological replicates per group) were subjected to immunoprecipitation with anti-hnRNPA2/B1 and anti-hnRNPA3 antibodies. The immunoprecipitates were resolved on 10% acrylamide gels, and Western blotting was performed with anti-phospho^Ser/Thr/Tyr antibody. (f, g) Densitometry analyses for phosphorylated hnRNPA2/B1 and hnRNPA3 levels, normalized to total hnRNPA2/B1 and hnRNPA3 levels, in M0, M1, and M2 Mφ are shown. ANOVA with Tukey’s post hoc test was performed to evaluate the significance presented in bar graphs ( value ≤ 0.05, M1 or M2 vs. M0).