Reduced HJV protein level and altered HJV cellular distribution in neogenin mutant mice. (A) Western blot analysis of HJV protein in different tissue lysates derived from P25 neogenin^+/+ and neogenin^m/m mice. (B) RT-PCR analysis to assess expression of transcripts for HJV from P20 muscles of neogenin^+/+ and neogenin^m/m mice. (C-D) Neogenin increases HJV protein level/stability. HEK293 cells were transfected Flag-HJV with or without neogenin. Cells were treated with CHX (an inhibitor of protein synthesis; 100 μg/mL) for indicated times. Lysates were probed with indicated antibodies. Blots were scanned with an Epson scanner and analyzed by National Institutes of Health ImageJ software. Quantitative analysis is shown in panel D. (E) Confocal images of coimmunohistochemical staining analysis of HJV protein distribution in wild-type and neogenin mutant liver at P25. Samples from 3 different mice were examined, and representative images examined by confocal microscope (Zeiss LSM 510 META, 40×/1.00 oil DIC) and processed using Adobe Photoshop CS2 software are shown. HJV (rabbit polyclonal, green), GS (monoclonal, red), and Topro3 (to label nuclei, blue) are indicated. The boxed areas were magnified and are shown in the bottom panels. The scale bars represent 100 μm. (F) Quantification analysis of data from panel E. The HJV immunoflourescence intensity per GS-positive cell is presented as means ± SEM (10 images from 3 different animals). *P < .01 compared with the wild-type liver.