Fig. 2
Reduced C/EBPβ protein in LysMCre-C/EBPβfl/fl microglia in culture. a Representative Western blot showing C/EBPβ LAP protein levels in primary microglial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice treated with vehicle (C) or LPS (100 ng/mL) + IFNγ (1 ng/mL) (L + I) for 24 h. Detection of the C/EBPβ isoforms Full and LIP in these samples required longer exposure. b Quantification of Western blot signals from four independent experiments as that shown in a. C/EBPβ protein levels are normalized using β-actin levels. Data are shown as mean + SEM. Significant effect of treatment is observed in C/EBPβfl/fl microglia (**p < 0.01), and significant effect of genotype is observed both in vehicle- and LPS + IFNγ-treated microglia (###p < 0.001). c Primary microglial cultures were treated as in a, fixed and C/EBPβ protein was analyzed by immunocytochemistry and nuclei were counterstained with DAPI. Note the nuclear C/EBPβ expression in virtually all microglial cells in C/EBPβfl/fl cultures which is enhanced by LPS + IFNγ and the complete absence of C/EBPβ immunoreactivity in LysMCre-C/EBPβfl/fl microglial cells in culture. Scale bar 100 μm. d, e Primary mixed glial cultures containing mainly astrocytes and microglia were treated as in a, fixed, immunostained for C/EBPβ (red) and the astroglial marker GFAP (green in d) or microglial marker Iba1 (green in e) and counterstained with DAPI. In C/EBPβfl/fl cultures, C/EBPβ immunostaining is observed in both GFAP-positive cells (astrocytes; arrows) and Iba1-positive cells (microglia; arrowheads), whereas in LysMCre-C/EBPβfl/fl cultures, C/EBPβ is only observed in astrocytes. Scale bar 50 μm