Fig. 2

a Wild-type and mutant IRP1 were inserted into the exon trapping vector pSplice Express using the attL1 and attL2 sites. The inserted fragment is flanked by exon a (exa) and exon b (exb), which are two constitutive insulin exons from rat. b PCR products (indicated by arrow) were amplified from cDNA generated from Hela cells using primer 1 and 2, to analyze the splicing reporter. c Sanger sequencing results of cDNA generated from mutant minigene showed a frameshift mutation and premature stop codon. Wild type minigene was sequenced as the control. d Immunohistochemistry showed negative staining for IRP1 at C-terminal and positive staining for IRP1 at N-terminal, HIF2α, EPOR and EPO in tumor cells. Normal adrenal medulla (NAM) were used as normal control, and sporadic PHEO tumor was used as the wild type tumor control. e Regulation of erythropoiesis via IRP1/HIF2α/EPO pathway