Figure 3

CNTF treatment results in protein phosphorylation and dephosphorylation in murine microglia. Enriched murine microglia were treated with CNTF (10 ng/mL) for 20 minutes or left untreated. One hundred micrograms of protein lysate were separated by isoelectrofocusing and subsequent SDS/PAGE under reducing conditions. Gels were fixed, stained with SYPRO Ruby, destained and then scanned on a 9410 Typhoon Imager. Panel A shows SYPRO Ruby staining of untreated microglia, and Panel B shows SYPRO Ruby staining of CNTF-stimulated microglia. For phosphospecific-immunoblotting, duplicate gels were transferred to nitrocellulose membranes and probed with an antibody against phospho-tyrsoine/serine/threonine to detect phosphorylated proteins in both untreated (C) and CNTF-stimulated microglia (D). Spots which showed altered phosphorylation are depicted at higher power for untreated (E) and CNTF stimulated microglia (F). CNTF altered phosphorylation of several proteins. Mass spectral analyses identified that Spot 1 is the hemopoietic cell specific Lyn substrate-1 (gi:13938627) and Spots 2a, 2b and 2c are β-tubulin 5 (gi:7106439). Spots 3 and 4 were too low in quantity to be unidentified. Data are representative of 3 independent experiments.