Figure 3

Validation of expression of Tlr4 transcripts by Northern blot and quantitative real-time-PCR. The expression of Toll-like receptor Tlr4 transcripts is detected by Northern blot and quantitative real-time (qRT) polymerase chain reaction (PCR), and is regulated in mouse bone-marrow derived macrophages by interferon (IFN) and lipopolysaccharide (LPS). Primary BMMs from two mouse strains (BALB/c and C57Bl/6J) were prepared and maintained overnight in the presence or absence of Csf1 (1 × 10⁴ U/ml) before stimulation with IFN-γ (500 pg/ml) or LPS (10 ng/ml). (a) qRT-PCR expression of full Tlr4 (left) and truncated variant (right). Both transcripts were detected at higher levels in BALB/c BMMs. The expression of the truncated variant was 10-fold lower than the full-length variant in both mouse strains. Delta CT values were calculated from three replicates and normalized to a HPRT control primer set. (b) The expression of Tlr4 mRNA from C57Bl6/J BMM was detected by Northern blot using a ³²P-labeled probe encoding part of the leucine rich repeat (LRR) domain, common to both variant Tlr4 transcripts. The membrane was stripped and re-probed with 18S ribosomal RNA as a loading control. The expression of both variants was induced by IFN and LPS. (c) SMART predictions of the protein domains found in the full-length (top) and truncated (bottom) Tlr4 transcripts. The variant transcript retains a signal peptide and leucine-rich domain, but it lacks the transmembrane and cytoplasmic domains.