Transfection of tagged constructs validate the expression and translation initiation site prediction of alternative proteins detected by LC-MS/MS.

Top diagrams represent the constructs used to detect the co-expression of HA-tagged reference and GFP-tagged alternative proteins by western blot analyses of HeLa cell lysates. GFP is inserted before the alternative stop codon in frame with the AltORF. The black line represents a specific region of the endogenous mRNA. For AltORFs located in 5′UTRs of ZNF83 and SLC35A4, the constructs do not contain the RefORF since the insertion of GFP may prevent the expression of the downstream RefORF. For AltORFs overlapping the 5′UTR and the RefORF (IDH3B), and for AltORFs overlapping the RefORF (BDH2, NIPA1, SCARB2), the HA tag was introduced before the GFP tag in frame with the RefORFs. Western blots show the co-expression of reference and corresponding alternative proteins in cell lysates with anti-HA and anti-GFP antibodies, respectively. The left and right lanes are cell lysates from cells expressing a construct with a normal alternative initiation AUG codon or with an inactivated alternative initiation AAG codon, respectively. NPTII, encoded in the expression plasmid, was used as a transfection control. Molecular weight markers in kDa are indicated on the right. Bottom panels show confocal/DIC images with the various cellular distributions of GFP-tagged alternative proteins. Nuclei were stained with Hoechst. Scale bar: 10 μm. * The reference protein was not detected due to the small size (