Figure 2
Characterization of A549 and HEK293 cell lines. (A) Duplicate samples of total RNA was extracted from 1 × 106 HEK293 (HEK) and A549 cells, reverse transcribed into cDNA and used as a template in PCR reactions using TLR4, Mal, CD14, MD2 and GAPDH gene-specific primers. Products were electrophoresed in 1.5% TBE agarose gels containing 0.5 μg/ml ethidium bromide and visualized under UV. (B) Western blot analysis of membrane (mem) and cytosolic (cyt) extracts (10 μg) from A549 and HEK293 cells probed with an anti-TLR4 antibody. Data are representative of three separate experiments. (C) For fluorescence microscopy, A549 cells (2 × 104) were grown in chamber slides, Fc-blocked and labelled with anti-TLR4 (clear) or isotype control antibodies (solid) and fluorophore-conjugated detection antibodies. TLR4 expression was quantified by laser scanning cytometry.