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Figure 3 | Journal of Neuroinflammation

Figure 3

From: The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity

Figure 3

GLT-1 expression is down-regulated by MIP-2γ in primary cortical astrocytes. (a) Levels of GLT-1 mRNA were decreased in primary astrocytes transfected with pAAV-MIP-2γ-hrGFP plasmids (MIP-2γ) compared with untreated cultures (control) or transfected with pAAV-IRES-hrGFP (mock) vector. MIP-2γ knockdown with siRNA-1 (MIP-2γ + siRNA-1), but not with pBS/U6-con (MIP-2γ + BS/U6-con) inhibits this down-regulation of GLT-1. No significant changes in GLAST, GFAP or β-actin occurred. (b) Representative western blot probed for GLT-1, GLAST, GFAP, and β-actin simultaneously. Levels of GLT-1 in astrocytes transfected with pAAV-MIP-2γ-hrGFP plasmids (MIP-2γ) decreased compared with untreated cultures (control) or transfected with pAAV-IRES-hrGFP (mock) vector, which could be partly reversed in astrocytes co-transfected with siRNA-1 (MIP-2γ + siRNA-1) but not with pBS/U6-con (MIP-2γ + BS/U6-con). Levels of GLAST and GFAP were unaffected in all groups. To ensure equal protein loading, blots were reprobed with β-actin-specific antibodies. (c) FACS analysis of GLAST or GLT-1 surface expression on astrocytes. Binding of GLAST or GLT-1 (shadow) compared with that of a rat isotype control IgG (dashed line) to intact untreated cultures (control), transfected with pAAV-IRES-hrGFP (mock) vector, transfected with pAAV-MIP-2γ-hrGFP plasmids (MIP-2γ), or pAAV-MIP-2γ-hrGFP plasmids co-transfected with siRNA-1 (MIP-2γ + siRNA-1) or pBS/U6-con (MIP-2γ + BS/U6-con) astrocytes. FACS, fluorescence-activated cell sorting; GFAP, glial fibrillary acidic protein; GLAST, glutamate-aspartate transporter; GLT-1, glutamate transporter; IgG, immunoglobulin G; MIP-2γ, macrophage inflammatory protein-2γ; siRNA, small interfering RNA.

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