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. 2012 Mar 12;7(3):e33140. doi: 10.1371/journal.pone.0033140

Figure 5. The p58 isoform of IGF2BP2 is generated by leaky ribosomal scanning.

Figure 5

A. A schematic representation of mIGF2BP2 mutants. Only the sequences surrounding the canonical translation initiation site and the internal Met69 are shown. The two putative translation initiation sites are in red. Cds, wild-type coding sequence of IGF2BP2; UTR, wild-type IGF2BP2 with 34 nt 5′UTR and 336 nt 3′UTR sequences included; Kozak1/2, wild-type IGF2BP2 with added Kozak sequence upstream of the canonical starting codon, 1/2Ile, the first and second ATG codon was mutated to ATC; 69Ile, the internal Met69 codon was mutated to ATC, mutKozak69, the Kozak consensus sequence flanking the internal Met69 was mutated; Δ5′, the first 66 codons including the first and second ATG codons were deleted. B. Protein analysis of mouse IGF2BP2 mutants expressed in 3T3-L1 preadipocytes (upper panels) and NIH-3T3 fibroblasts (lower panels) using retrovirus vectors. Untreated cells, and cells transfected with the empty vector pBabe, were used as controls. Cellular proteins (10 µg/lane) were resolved by 10% SDS-PAGE and blots were probed with antibody to IGF2BP2 C-terminal peptide (CRB Ltd) and with antibody to the p85 subunit of PI3-kinase as a loading control.