Fig. 3

The short SOCS3 isoform is more stable than the long isoform of SOCS3. a HEK293 cells were transfected with expression vectors for SOCS3 M12 V or SOCS3 dN. Cells were treated with 50 μg/mL cycloheximide for the times indicated. SOCS3 and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. Lower panel: quantification of relative SOCS3 protein expression of the long (blue) and short SOCS3 (red) isoform. Expression was normalized to tubulin expression. Maximal SOCS3 protein expression was set to 100%. Representative results of n = 4 independent experiments are shown (Additional file 1: Figure S3). b HEK293 FlpIn cells stably transfected to express doxycycline-dependently SOCS3 pre-mRNA were treated with 1 μg/mL doxycycline for 4 h to induce expression of the SOCS3 isoforms. After withdrawal of doxycycline, cells were treated with MG132 (10 μg/mL). After 15 min 50 μg/mL cycloheximide was added for the times indicated. SOCS3 and tubulin expression were evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. Representative results of n = 3 independent experiments are shown. For quantification see Additional file 1: Figure S4. c Quantification of the half-lives of the long (blue) and short SOCS3 (red) isoform in the absence or presence of MG132 from experiments as shown in Fig. 3b. Expression was normalized to tubulin expression. Maximal SOCS3 protein expression was set to 100% for each specific data set. Calculation of the half-lives was performed by linear regression for each specific data set. Data are given as mean of three independent experiments ± SD. Half-lives of the long and short SOCS3 isoform differ significantly in the absence of MG132 treatment (rANOVA: * p = 0.016) but are equal in the presence of MG132 (rANOVA: n.s. p > 0.05)