Figure 3.

Pa lung infection increases NEU1-MUC1 and PPCA-MUC1 association and MUC1-ED desialylation. A–E and G–J, BALB/c mice were administered i.n. with 1.0 × 10⁵ CFUs/mouse of PAK or the PBS vehicle. At 24 h postchallenge, BALF and lung tissues were collected, and lungs were homogenized. A, lung homogenates were immunoprecipitated with anti-MUC1-CD (lanes 1, 2, 5, 6), anti-NEU1 (lanes 3, 4), or anti-PPCA (lanes 7, 8) Abs. The MUC1-CD immunoprecipitates were processed for NEU1 (lanes 1, 2) or PPCA (lanes 5, 6) immunoblotting, and the NEU1 (lanes 3, 4) and PPCA (lanes 7, 8) immunoprecipitates were processed for MUC1-CD immunoblotting (upper panels). To control for protein loading and transfer, the immunoblots were stripped and reprobed with the immunoprecipitating Ab (lower panels). B–E, densitometric analyses of the blots in (A). Error bars represent mean ± S.E. MUC1-CD, NEU1, or PPCA signal normalized to the immunoprecipitate signal in the same lane on the same stripped and reprobed blot (n = 3). *, increased normalized lung NEU1, MUC1-CD, or PPCA signal compared with PBS controls at p < 0.05. F, to validate PNA selectivity, the negative control, fetuin (lane 1), and the positive control, asialofetuin (lane 2), 1.0 μg each, were processed for PNA lectin blotting. G, BALF (lanes 1 and 2) and lung homogenates (lanes 3 and 4) from mice administered i.n. with PAK or the PBS vehicle were processed for MUC1-ED immunoblotting. H, BALF (lanes 1 and 2) and lung homogenates (lanes 3 and 4) were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. I and J, densitometric analyses of the blots in (H). Error bars represent mean ± S.E. BALF/lung desialylated MUC1-ED signal (n = 3). *, increased BALF MUC1-ED signal compared with PBS controls at p < 0.05. (A and F–H), molecular masses in kDa are indicated on the left. IP, immunoprecipitate; IB, immunoblot; IB*, immunoblot after stripping. PD, pulldown. The results are representative of three independent experiments.