Generation of hepatocyte-specific conditional Neo1 knockout (Neo1^fl/fl;Alb-Cre⁺) mice. (A) Diagram of NEO1 and the domains that HJV/RGMa/RGMb bind. FNIII, fibronectin III; Ig, immunoglobulin; PM, plasma membrane. (B) Schematic depictions of loxP-flanked (floxed) Neo1 allele and the allele after Cre recombinase-mediated excision. Genotyping primer positions are indicated. (C) PCR analysis of genomic DNA extracted from the liver and intestine of wild-type (WT), Neo1^fl/fl;Alb-Cre⁻, and Neo1^fl/fl;Alb-Cre⁺ mice. (D) qRT-PCR analysis of Neo1 mRNA levels in the liver and isolated hepatocytes from 8-week-old WT, Neo1^fl/fl;Alb-Cre⁻, Neo1^fl/fl;Alb-Cre⁺, or Neo1^fl/wt;Alb-Cr⁺ mice. Each group consists of at least 3 animals. Data shown are means ± SD. **P < .01 and ***P < .001 relative to the respective Neo1^fl/fl;Alb-Cre⁻ controls. (E-F) Representative images of western blot analysis for Neo1 and β-actin in the liver (E) and isolated hepatocytes (F). Two images of Neo1 with different exposure times were presented. IB, immunoblotting.