Figure 4.
Figure 4. Functional studies of mutations in VHL E1′. (A-B) Immunoblot analysis of patient LCLs. A representative immunoblot (A) and quantification of 3 different immunoblots (B) are presented. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with LCL control (C1) were fixed at 100%. Data are represented as the mean ± standard error of the mean. A VHL antibody that recognized VHL E1 was able to detect the hypothetical X1 protein. In the most left lane, control LCLs were transfected with an expression vector containing the coding sequence for X1; 5 µg of proteins were blotted vs 45 µg for other samples to avoid signal saturation with overexpressed X1. Patients with erythrocytosis are indicated in red. (C) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells (ie, VHL− cells that constitutively express HIF-2α). The results are expressed as relative firefly luciferase activity with Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. The ability of WT and mutated X1 to downregulate firefly luciferase activity (related to HIF activity) was compared with pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag (HA) was used to detect HA-VHL and HA-X1. X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G mutations on the hypothetical X1 protein. Three independent experiments were performed. (D) Characterization of VHL E1′ retention by the minigene experiment (representative picture of agarose gel; n = 3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E1′ (WT or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected, and the expression of the spliced chimeric transcripts (containing EA and EB ± E1′) was analyzed. Two WT constructs containing E1′ were used; 1 contained the single-nucleotide polymorphism c.340+1150T>C (rs779808). Bands corresponding to EA and EB spliced together or with VHL E1′ are indicated on the right. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. †Corresponds to unspecific bands verified by sequencing. The minigene experiment performed with the construction carrying the mutation c.340+574A>T (that targets the SA site of E1′ in F6) confirmed the use of an alternative SA site (right panel) with the deletion of 15 nucleotides (represented in red). ns, not significant.

Functional studies of mutations in VHL E1′. (A-B) Immunoblot analysis of patient LCLs. A representative immunoblot (A) and quantification of 3 different immunoblots (B) are presented. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with LCL control (C1) were fixed at 100%. Data are represented as the mean ± standard error of the mean. A VHL antibody that recognized VHL E1 was able to detect the hypothetical X1 protein. In the most left lane, control LCLs were transfected with an expression vector containing the coding sequence for X1; 5 µg of proteins were blotted vs 45 µg for other samples to avoid signal saturation with overexpressed X1. Patients with erythrocytosis are indicated in red. (C) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells (ie, VHL− cells that constitutively express HIF-2α). The results are expressed as relative firefly luciferase activity with Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. The ability of WT and mutated X1 to downregulate firefly luciferase activity (related to HIF activity) was compared with pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag (HA) was used to detect HA-VHL and HA-X1. X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G mutations on the hypothetical X1 protein. Three independent experiments were performed. (D) Characterization of VHL E1′ retention by the minigene experiment (representative picture of agarose gel; n = 3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E1′ (WT or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected, and the expression of the spliced chimeric transcripts (containing EA and EB ± E1′) was analyzed. Two WT constructs containing E1′ were used; 1 contained the single-nucleotide polymorphism c.340+1150T>C (rs779808). Bands corresponding to EA and EB spliced together or with VHL E1′ are indicated on the right. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. †Corresponds to unspecific bands verified by sequencing. The minigene experiment performed with the construction carrying the mutation c.340+574A>T (that targets the SA site of E1′ in F6) confirmed the use of an alternative SA site (right panel) with the deletion of 15 nucleotides (represented in red). ns, not significant.

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