Figure 3.

The uORF of SCL controls translation initiation at alternative initiation sites. COS-1 cells were transiently transfected with SCL constructs and analyzed by immunoblotting using HA-epitope-specific antiserum. (A) The wild-type uORF initiation site (wtΦ) was compared to an optimized initiation context (Φ⁺), a weakened context (Φ⁻), or a noninitiation site (ΔΦ). (B) Mutation of alternative translation initiation sites affects SCL-isoform expression. Null (Δ) mutations of the individual initiation sites (A, B, or C) are shown. X indicates an additional initiation site engineered between sites B and C with the uORF initiation codon in wild-type context (X) or removed (XΔΦ). Schematic representations of the SCL mRNA with initiation sites indicated in relation to the protein bands is shown at the left. An alternative CUG initiation codon is depicted with an arrowhead (▹).