HJV-mediated BMP signaling and hepcidin expression are not altered by neogenin overexpression in Hep3B cells. (A) Hep3B cells were transfected with BRE-Luc and pRL-TK, either alone or with a fixed amount of Flag-HJV cDNA in combination with increasing amounts of HA-neogenin cDNA for 46 hours. Cell lysates were analyzed for luciferase activity as in Figure 1B-E (top panel) or by Western blot in succession with Flag antibody (M5; α-Flag), neogenin antibody (α-NEN), and actin antibody (α-actin, as a loading control; bottom panel). (B) Hep3B cells were transfected with BRE-Luc and pRL-TK, either alone or with fixed amount of HA-neogenin cDNA (40 ng/mL), in combination with increasing amounts of Flag-HJV cDNA. Cell lysates were analyzed for luciferase activity as in Figure 1B-E. (C,D) Hep3B cells were transfected with Hep-Luc and pRL-TK, either alone or with Flag-HJV at 2 ng/mL (C) or 8 ng/mL (D), in combination with increasing amounts of HA-neogenin cDNA. Cell lysates were analyzed for luciferase activity as in Figure 1B-E. (E) Hep3B cells were transfected with empty vector (pcDNA3) or Flag-HJV cDNA in combination with increasing amount of HA-neogenin cDNA. After 46 hours, hepcidin relative to RPL19 mRNA levels were quantified by real-time RT-PCR as in Figure 1G,H. Values shown are the means of triplicate measurements plus or minus SD.