Hrp tagged goat anti mouse and anti rabbit igg

For protein analysis, GL261 cells (2 × 10⁵) were seeded on 12 well plates and treated with GlaB 5 μm for different times (5–10–20-30 min), washed with PBS and lysed in hot 2x Laemmli buffer, boiled 5 min and sonicated. The same amount of proteins was separated on 10% SDS-polyacrylamide gel electrophoresis and analyzed by western immunoblot using the following primary antibodies: P-AMPKα (1:2500, Cell Signaling), AMPKα (1:2000, Cell Signaling); HRP-tagged goat anti-mouse and anti-rabbit IgG were used as a secondary antibody (1:2000; Dako). Detection was performed through the chemiluminescent assay Immun-Star Western C Kit (Bio-Rad, CA) and densitometric analysis was carried out with Quantity One software (Bio-Rad, CA).

For protein analysis, microglial cells were seeded on 12 well plates (40 × 10⁴ cells) and treated with LPS (100 ng/ml) for 24 h and the day after with CX3CL1 (100 nM) for further 24 h; cells were washed with PBS and lysed in hot 2× Laemmli buffer, boiled 5 min and sonicated. The same amount of protein samples was separated on 12% SDS-polyacrylamide gel electrophoresis and analyzed by western immunoblot using the following primary antibodies: Arg-1 (1:200, Santa Cruz Biotechnology) PKM2 (1:2000, Cell Signaling), Actin (1:2000 Sigma-Aldrich), HRP-tagged goat anti-mouse and anti-rabbit IgG were used as a secondary antibody (1:2000; Dako). For protein analysis we used four different primary microglial culture preparations. Detection was performed through the chemiluminescent assay Immun-Star Western C Kit (Bio-Rad, CA) and densitometric analysis was carried out with Quantity One software (Bio-Rad, CA).

For protein analysis, cells were seeded on 24 well plates (6 × 10⁵ cells); cells were washed with PBS and lysed in hot 2× Laemmli buffer, boiled 5 min and sonicated. The same amount of proteins was separated on 8.75% SDS-polyacrylamide gel electrophoresis and analyzed by western immunoblot using the following primary antibodies: E-Cadherin (1:200, Santa Cruz Biotechnology) N-Cadherin (1:2000, Millipore), EGFR (1:1000, Cell Signaling), beta-catenin (1:2000, Sigma-Aldrich), actin (1:5000, Sigma-Aldrich); HRP-tagged goat anti-mouse and anti-rabbit IgG were used as a secondary antibody (1:2000; Dako). Detection was performed through the chemiluminescent assay Immun-Star Western C Kit (Bio-Rad, CA) and densitometric analysis was carried out with Quantity One software (Bio-Rad, CA).