Fig. 3: Created uAUGs in the 5’UTR of ENG seem to be able to initiate the translation.

a cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies^2,21,22,23 as well as position of the associated uStop codon (c.125), the introduced deletion c.1_2del, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR 5’ untranslated region, CDS coding sequence, WT wild type, uoORF overlapping upstream open reading frame. b Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers¹⁶. Anti-Myc and anti-βactin correspond to the used antibodies for the target and the reference proteins, respectively. kDa kilodalton, M protein ladder, WT wild type, C- negative control corresponding to pcDNA3.1- empty vector. Shown results are representative of 5 independent experiments. Uncropped blots are shown in Supplementary Fig. 4. All blots were processed in parallel and derive from the same experiments. c Quantification of protein steady-state levels obtained in (b) and probably resulting from translation initiation at predicted uAUGs. For quantification, the average of each duplicate has been calculated from the quantified values and ENG levels for each sample have been normalized to the corresponding β-actin levels then to the WT (%). na, no 5’UTR variant introduced. Graphs with standard error of the mean are shown and are representative of 5 independent experiments. ***p value < 10⁻³; **p value < 10⁻²; *p value < 5 × 10⁻², ns non-significant (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).