Gene targeting strategy for B-hTWEAK/hAPRIL mice. The full sequence of mouse Tnfsf12 gene and mouse Tnfsf13 gene were replaced with the corresponding human sequences in B-hTWEAK/hAPRIL mice.

Strain specific analysis of TWEAK and APRIL gene expression in wild type (WT) mice and B-hTWEAK/hAPRIL mice by RT-PCR. Mouse Tweak and April mRNA were detectable only in lung of WT mice (+/+). Human TWEAK and APRIL mRNA was detectable only in homozygous B-hTWEAK/hAPRIL mice (H/H) but not in WT mice (+/+).

Strain-specific APRIL expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTWEAK/hAPRIL mice by ELISA. Serum were collected from wild-type C57BL/6JNifdc mice (+/+) (n=3, 7 weeks old, male) and homozygous B-hTWEAK/hAPRIL mice (H/H) (n=3, 7 weeks old, male) stimulated with LPS in vivo (200 μg/mouse, stimulation for 4 hours, i.p.). Expression level of human APRIL were analyzed by ELISA (human APRIL ELISA kit: thermofisher, BMS2008). Human APRIL was exclusively detectable in homozygous B-hTWEAK/hAPRIL mice, but not in wild-type C57BL/6JNifdc mice. Values are expressed as mean ± SEM.

Strain specific TWEAK expression analysis in homozygous B-hTWEAK/APRIL mice by western blot. Lung was collected from wild type (WT) mice (+/+) and homozygous B-hTWEAK/APRIL mice (H/H), and analyzed by western blot with anti-TWEAK antibody. TWEAK was detectable in WT mice (+/+) and homozygous B-hTWEAK/APRIL mice (H/H) due to the cross-reactivity of antibodies.