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Fig. 6. Synthesis of LN-5 by TECs. (A) RT-PCR analysis of isolated primary TECs (PTEC) and CD11c+ dendritic cells (CD11c+) showed that only primary TECs synthesize the 
3, ß3 and 
2 chains of LN-5. Total RNA was extracted from both cell types and from the lung adenocarcinoma cell line A549, which served as a positive control. After reverse transcription, cDNA quality was checked by ß-actin PCR. Equal amounts of cDNA were subjected to PCR analysis for the laminin 3, ß3 and 2 chains. Whereas the control cell line A549 and primary TECs synthesize all three chains of LN-5, dendritic cells do not. (B) By immunoprecipitation of TEC lysates with antibodies against the laminin 3 and 2 chain, and subsequent immunoblotting (WB) of the immunoprecipitates (IP) with the anti-laminin 2 chain (LN-2-chain) antibody, the processed form of laminin 2 chain at 105 kDa could be detected. Co-precipitation of the laminin 3 chain with laminin 2 revealed an intact LN-5 isoform. The bands at 55 kDa represent the heavy chains of the precipitating antibodies. (C,D) Immunofluorescence staining of TECs cultured in vitro with the laminin 2 chain antibody showed a strong staining signal in red, the cell nuclei are counterstained with DAPI in blue (D). Dendritic cells, however, did not synthesize LN-5 since no labeling of the dendritic cells in vitro could be detected with the laminin ß3 chain antibody, only the DAPI counterstaining of the nuclei is visible (C). Bar, 15 µm.
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