Adaptive Immunity, Autophagy Figure 4. CD40 upregulates autophagy protein expression and leads to increased autophagy. A, Primary brain microglia from B6 mice were incubated with anti-CD40 mAb or control mAb. Beclin 1, Atg5 and Atg7 protein expression were normalized against actin. B, Beclin 1 and actin protein expression in purified brain microglia from uninfected B6 and CD402/2 mice or purified brain microglia/macrophages from B6 or CD402/2 mice at 4 weeks post-infection. Each lane represents a pool of 2 mice. C, D, BV-2 cells transfected with LC3-EGFP were incubated with anti-CD40 mAb or control mAb in the presence or absence of bafilomycin A1 (BafA1). E, BV-2 cells were transfected with control or Beclin 1 siRNA. After 2 days, cells were transfected with LC3-EGFP followed by incubation with anti-CD40 or control mAb. F, BV-2 cells were transfected with Beclin 1-encoding or empty plasmids. Expression of Beclin 1 and actin were examined by immunoblot. Cells were incubated with anti-CD40 mAb (1 or 10 mg/ml) or control mAb followed by infection with tachyzoites of RH T. gondii. G, BV-2 cells transfected with a suboptimal concentration of Beclin 1 siRNA or control were incubated with anti-CD40 mAb (10 mg/ml) or control mAb followed by RH T. gondii infection. Results are representative of 3 to 4 independent experiments. **p#0.01; ***p#0.001. doi:10.1371/journal.pone.0014472.g004 PLoS ONE | www.plosone.org 6 December 2010 | Volume 5 | Issue 12 | e14472