Figure 2.

Differential start codon usage of the TSC22D1.2 transcript. (A) Immunoblot for TSC22D1.2 of HDF with or without BRAF^E600, 8 days after BRAF^E600 expression. β-Actin serves as a loading control. (B) To express TSC22D1.2, three different constructs were created. TSC22D1.2a has an optimal Kozak sequence in front of the first ATG to initiate translation of a 144-aa protein; TSC22D1.2b contains the nine base pairs of the genomic locus in front of the first ATG, enabeling the start of translation similar to the wild-type situation; in TSC22D1.2c the second ATG is mutated to TTG, leading to the expression mainly of an 86-aa protein. All three variants can be depleted by specific shRNAs, confirming the identity of the proteins (see Supplementary Figure S8). Immunoblot for TSC22D1.2 of HDF transduced with the various TSC22D1.2 constructs. CDK4 serves as a loading control. (C) Schematic drawing of the TSC22D1.2 protein. The TSC box, a highly conserved domain found in all TSC22 protein family members, is depicted in blue. The grey box indicates the leucine zipper domain. Arrowheads indicate the translational start sites of the 144-aa (red), 134-aa (green) and 86-aa (black) polypeptides along the TSC22D1.2 transcript.