Fig. 2. Cellular expression of anisoforms.

a, Schematic of iORFs and CDSs in canonical (upper panel) and alternative transcripts (lower panel) for four representative genes. b, Alternative transcripts from the 5’ UTR to the 3’ end of the iORF, which was fused with a HA-FLAG tag at the C-terminus in an expression vector. Overexpression in HEK 293T cells was followed by anti-FLAG Western blotting. The CENPM2 anisoform exhibits aberrant mobility in SDS-PAGE. c, Anisoforms overexpressed in HEK 293T from the cDNA clones described in (b) were subjected to anti-FLAG immunofluorescence (green) with DAPI counterstain (blue). Scale bars, 20 μm. d, Schematic of FLAG knock-in (KI) strategy to validate endogenous anisoform expression in cells. The HEK293T cell genome was cleaved by Cas9 and sgRNAs targeting the stop codons of two independent iORFs (DEDD2 and RUSC1). Single-stranded oligodeoxynucleotide (ssODN) templates encoding FLAG tags were then used to guide homology-directed repair (HDR). Editing in FLAG KI cells was validated with PCR (Extended Data Fig. 5c). e, Western blotting of anti-FLAG immunoprecipitates exhibited specific signal corresponding to anisoform expression in clonally selected KI cell lines relative with parental HEK 293T (WT) as a control. The DEDD2 anisoform-FLAG shows two bands, which may arise from alternative splicing.