Fig 1: NanA dysregulated the interaction of Siglec-5 with TLR2 and SHP-1. (A) Surface desialylation analysis of WT SPN- and ?nanA-infected THP-1 cells. THP-1 cells were infected with CFSE-labeled pneumococcus (green) at an MOI of 30 for 1 h followed by staining with biotin-conjugated ECA and Alexa Fluor 568-conjugated streptavidin (red). DNA was stained with DAPI (blue). (B) The distribution of TLR-2 and Siglec-5 in the lipid rafts. Sig-5/THP-1 cells were uninfected (Un) or infected with WT SPN and ?nanA at MOI of 10 for 1 h. Cell lysates collected from infected cells were separated by sucrose-gradient ultracentrifugation and immunoblotting with Ab recognizing TLR2, Siglec-5, SHP-1, SHP-2, and flotillin-1. The summed band intensities of TLR-2 and Siglec-5 in the raft fractions 4 and 5 were quantified by Image J. (C) Sig-5/THP-1 cells were infected with WT SPN and ?nanA at an MOI of 10 for 1 h. The sialylation level and Siglec-5-interaction of TLR-2 were analyzed by immunoprecipitating cell lysates with anti-TLR-2 Ab, followed by probing with PNA and anti-Siglec-5 Ab, respectively. Band intensities of PNA signal and co-precipitated Siglec-5 were quantified by Image J and normalized to TLR-2 values. (D) Sig-5/THP-1 cells were infected with WT SPN and ?nanA at an MOI of 10 for 40 min and the SHP-1 recruitment to Siglec-5 was examined by immunoprecipitating cell lysates with anti-Siglec-5 Ab, followed by probing with anti-SHP-1 Ab. Band intensities of co-precipitated SHP-1 were measured by Image J and normalized Siglec-5 values. The data shown in Figure 2 were representative of two independent experiments.