Figure 4.

Both MyD88-dependent and TRIF-dependent signaling pathways increase MyD88-S production. A and B, MyD88-L and MyD88-S expression in RAW264.7 cell lines stably expressing either CAT, MyD88-GyrB, or TRAF6-GyrB. The cell lines were either treated with coumermycin A1 (CM), which dimerizes the gyrB fusions, resulting in activation of that protein, or not treated (NT) for 24 h prior to RNA collection for qPCR. C, TNFα protein production in the supernatants (monitored by ELISA) from the studies in A and B. D–F, plasmids overexpressing either negative control protein CAT or TRIF were transiently transfected into RAW264.7 cells; 48 h after transfection, MyD88-L and MyD88-S mRNA levels were assessed by qPCR in conjunction with isoform-specific primers, or TNFα protein production was monitored by ELISA. All experiments represent a minimum of three independent biological replicates. Data are normalized so that MyD88-L or MyD88-S expression in the absence of LPS is set to 1. *, p < 0.05.