Co-expression of alternative and reference proteins in cDNA transfection experiments is common.

(A) Distribution of the number of predicted RefORFs-contained AltORFs per gene in the human genome. Top, schematic representation of a mRNA with a RefORF (grey)-containing AltORF (green). By definition, RefORFs are present in the +1 reading frame and AltORFs are present in the non-canonical +2 and +3 reading frames. (B) Strategy to detect the co-expression of reference and alternative proteins in cDNA transfection experiments. HA and GFP tags permit the detection of reference and alternative proteins, respectively. Top, graphical representation of a mRNA with a RefORF-contained AltORF. Middle, typical cDNA construct used in transfection experiments. Bottom, representation of constructs used in (C). (C) Western blot analyses of HA-tagged LGALS3BP (Lectin galactoside-binding soluble 3 binding protein), VEGFC (vascular endothelium growth factor), p53 (cellular tumor antigen p53), CDC42 (cell division cycle 42), BDKRB2 (bradykinin receptor), and SRSF1 (serine/arginine-rich splicing factor 1), and their respective GFP-tagged alternative proteins using anti-HA and anti-GFP antibodies (top panels). Bottom panels show the cellular distribution of alternative proteins by confocal fluorescence microscopy (differential interference contrast and Hoechst, left panels; GFP, right panels). Scale bar: 10 μm.