Fig. 4
The UTRs and the first AUG codon influence the ratio of SOCS3 isoform expression. a Schema of the SOCS3 constructs analyzed in the following experiments (orange: coding sequence; grey: intron; blue: UTR; green: alternative translational start sites). P1 and P2 represent the primer pair used for amplification and discrimination of non-spliced SOCS3 pre-mRNA and spliced SOCS3 mRNA. For information on the Kozak sequence of the specific constructs see Additional file 1: Figure S1) (b) A specific primer pair corresponding to sequences located 5′ and 3′ to the single intron was used to discriminate spliced mRNA and un-spliced pre-mRNA (see Fig. 4a). For reference genomic DNA from untreated HepG2 cells was isolated and a DNA fragment corresponding to the unspliced pre-mRNA of SOCS3 was amplified (lane 1). Expression vectors for SOCS3 mRNA and SOCS3 pre-mRNA (Fig. 4a) were used as templates for amplification of DNA fragments corresponding to SOCS3 mRNA or unspliced SOCS3 pre-mRNA, respectively (lanes 2 and 3). Amplification products were separated by agarose gel electrophoresis. c HepG2 cells were stimulated with 10 ng/mL IL-6 for the indicated times. mRNA was isolated, translated into cDNA and cDNA amplified as in Fig. 4b. Amplification products were separated on the same gel by gel electrophoresis. The dashed line indicates removed lanes. Exposure was identical to all parts of the gel. Amplicons corresponding to spliced mRNA and non-spliced pre-mRNA are indicated by arrows. For quantification see Additional file 1: Figure S5. d HEK293 cells were transfected with expression vectors for SOCS3 pre-mRNA, SOCS3 mRNA, the coding sequence of SOCS3 (SOCS3 cds), and the coding sequence of the short isoform of SOCS3 (SOCS3 dN) (Fig. 4a). SOCS3 and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. For quantification see Additional file 1: Figure S6. Representative results of n = 3 independent experiments are shown. For expression of the different SOCS3 mutants, we adjusted the amount of transfected DNA in accordance to vector size. e HEK293 cells were transfected with expression vectors for SOCS3 mRNA, SOCS3 mRNA d5’UTR, SOCS3 mRNA d5’UTR dKozak, and SOCS3 cds. SOCS3 and tubulin protein expression was evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. For expression of the different SOCS3 mutants we adjusted the amount of transfected DNA in accordance to vector size. f HEK293 cells were transfected with expression vectors containing the coding sequence of SOCS3 (lane 2; cds), expression vectors where the first AUG in the coding sequence of the long SOCS3 isoform was mutated (SOCS3 dAUG1) or expression vectors were the coding sequence of the long isoform-specific peptide was deleted (SOCS3 dN). SOCS3 and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. Representative results of n = 3 independent experiments are shown