Fig. 2.
Characterization of cardiomyocyte- and myeloid-specific MyD88 deletion mice. (A) Exon 3 of MyD88 gene in various tissues DNA from α-MHC-MyD88−/− or Lyz-MyD88−/− mice was amplified by polymerase chain reaction using specific primers. Upper band (950 base pairs) indicated the intact loxP-flanked MyD88 gene. Lower band (500 base pairs) indicated the deletion of exon 3 of MyD88 gene. (B) Transcript of MyD88 gene in cardiomyocyte (CM) or bone marrow–derived macrophage (Mϕ) isolated from α-MHC-MyD88−/− or Lyz-MyD88−/− mice was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Each error bar represents mean ± SD. ** P < 0.01, n = 3 in each group. (C) Western blot was used to detect MyD88 protein expression in CM or Mϕ isolated from α-MHC-MyD88−/− or Lyz-MyD88−/− mice. (D) Mϕ isolated from α-MHC-MyD88−/−, Lyz-MyD88−/−, MyD88fl/fl, and MyD88−/− mice were treated with 10 ng/ml of Pam3Cys (a TLR2 ligand activating TLR2 → MyD88 signaling) or 25 μg/ml of Poly(I:C) (a TLR3 ligand activating TLR3 → Trif signaling) (Enzo Life, Farmingdale, NY). Two hours later, the cells were harvested for tumor necrosis factor-α (TNFα) production as measured by qRT-PCR. Each error bar represents mean ± SD. *** P < 0.001, n = 3 in each group. del = deletion; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; Kid = kidney; Liv = liver; Lu = lung; Lyz-MyD88−/− = myeloid-specific MyD88 knockout mice; Mϕ = bone marrow–derived macrophage; (α-) MHC-MyD88−/− = cardiomyocyte-specific MyD88 knockout mice; α-MHC = α-myosin heavy chain; Mus = skeletal muscle; MyD88 = myeloid differentiation factor 88; MyD88−/− = MyD88 knockout mice; MyD88fl/fl = MyD88-loxP control mice; Pam3 = Pam3Cys; Spl = spleen; TLR = Toll-like receptor.