Fig 1: Validation of polySia expression and polysialylated proteins in BV2 microglia. a Immunofluorescence staining of polySia co-localized with the Golgi marker giantin. Cell shape is highlighted by co-staining with the microglia/macrophage marker CD11b. Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm. b Immunoprecipitation (IP) of polysialylated proteins from lysate of 107 BV2 cells using polySia-specific mAb 735-conjugated magnetic beads followed by Western blot (WB) detection with polySia-specific antibody (left), or by joint incubation with NRP2- and ESL-1-specific antibodies (right). Where indicated, IP fractions were treated with endosialidase (endo +), to remove polysialic acid. Protein bands were assigned according to the apparent molecular weights of NRP2 and ESL-1 as previously detected in primary and stem cell-derived murine microglia or in mouse brain tissue [5; see text for details]. c Compared to untreated controls (ctrl), incubation of BV2 cultures with 1-µg/ml LPS for 24 h leads to the loss of polySia signals in almost all cells. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. d Loss of polySia-positive cells after LPS treatment, as indicated. Individual values and means of 5 evaluated frames per culture condition, with a minimum of 20 cells each are plotted and significant difference by two-tailed t-test is indicated (*** p < 0.001). e PolySia with DP > 4 but not trisialic acid (DP3) attenuates the LPS-induced production of NO. Nitrite levels in the supernatant of BV2 cells cultured for 24 h in the presence or absence of 30-nM trisialic acid (DP3), 500-ng/ml poly—Sia (approximately 30 nM, see text for details), and/or 1-µg/ml LPS, as indicated. Individual values and means from 3 independent treatments per group are plotted. One-way ANOVA indicated significant differences (p < 0.0001) and results from Tukey’s post hoc test are shown for comparisons between the LPS-treated groups (*p < 0.05)