Figure 4

Protein expression of P-glycoprotein and Mrp1 in HAPI cells following 6 or 24 hour LPS incubation. (A) HAPI microglia were incubated for 6 or 24 hours in the presence of increasing concentrations of LPS (1 to 10 ng/ml). At the end of the incubation period, crude membrane fractions from the cell lysates or from rat kidney brush border membranes (positive P-glycoprotein control) were prepared as indicated in the text, and immunoblotting of P-glycoprotein was performed. The proteins (1 or 40 μg each) were separated on NuPage 7% sodium-acetate gels, transferred to polyvinylidene difluoride membranes electrophoretically and incubated with a P-glycoprotein monoclonal antibody, C219 (1:100), followed by an anti-mouse HRPO-secondary (1:1,000). (B) Immunoblotting of Mrp1 in HAPI microglia treated for 6 or 24 hours with increasing concentrations of LPS (1 to 10 ng/ml) was undertaken in a similar manner to P-glycoprotein except that the proteins (10 μg) were separated on NuPage 4 to 12% Bis-Tris gels, and incubated with a Mrp1 monoclonal antibody, MRPr1 (1:100), followed by an anti-rat HRPO-secondary (1:1,000). Whole rat kidney lysate (10 μg) was used as a positive Mrp1 control. Representative immunoblots are shown.