Western blot analysis of GFRA1 protein expression in homozygous B-hGFRA1 mice. Various tissue lysates were collected from wild-type C57BL/6 mice(+/+) and homozygous B-hGFRA1 mice (H/H), and then analyzed by western blot with anti-GFRA1 antibody. 40 μg total proteins were loaded for western blotting analysis. GFRA1 protein was detected in the heart, lung and brain of wild-type C57BL/6 mice and homozygous B-hGFRA1 mice, as the antibody is cross-recognize both human and mouse GFRA1. β-Actin is expressed at very low levels in the heart and undetectableand.

Strain specific analysis of GFRA1 mRNA expression in wild-type C57BL/6 mice and B-hGFRA1 mice by RT-PCR. Brain RNA was isolated from wild-type C57BL/6 mice(+/+) and homozygous B-hGFRA1 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GFRA1 primers. Mouse Gfra1 mRNA was detectable only in wild-type C57BL/6 mice. Human GFRA1 mRNA was detectable only in homozygous B-hGFRA1 mice but not in wild-type mice. And the PCR products were confirmed with Sanger sequencing.