Figure 2.

AHNAK1 is required for protection against Leishmania major infection. Wild-type and AHNAK1 mice were challenged in the right hind foot with 10⁶ L. major stationary phase promastigotes. (A) Lesion development (as a ratio of infected foot to non-infected foot) results with time postinfection. Results are representative of at least three independent experiments. (B) At 2 weeks postinfection, four mice per group were sacrificed and parasite burden was determined by limiting dilution assay. Results shown are representative of two independent experiments. (C) wild-type IFNγ and IL-4 levels were measured by ELISA in 96 hrs culture supernatant of purified popliteal lymph node CD4 T cells from infected mice cultured with irradiated wild-type APCs and indicated doses of L.major antigen. Results shown are representative of two independent experiments with 3 infected mice in each group. (D) Macrophages and CD4 T cells were isolated from wild-type mice. The expression of AHNAK1 in these cells was compared by western blot analysis using anti-AHNAK1-C2 antibody. AHNAK1^−/− CD4 T cell extract and β-actin were used as controls for the specificity of the AHNAK1 antibody and loading, respectively. (E) Peritoneal macrophages from wild-type and AHNAK1^−/− mice were infected with L. major in vitro and cultured for 72 hours in the either presence or absence of LPS (200 ng/ml) and IFNγ (300 ng/ml). Internalized parasites were detected by staining with DAPI and counted. Results are representative of at least three independent experiments.