Fig. 5

Neither PKR activation nor PKR knockout affect SOCS3 isoform expression. a HeLa cells were transfected with expression vectors encoding SOCS3 pre-mRNA or SOCS3 mRNA. After transfection cells were treated with 10 μg/mL pIC for 3 h. PKR phosphorylation ((p)T451), eIF2α phosphorylation ((p)S51) and PKR, eIF2α, SOCS3, and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoform of SOCS3 respectively. Representative results of n = 3 independent experiments are shown. b HEK293 FlpIn SOCS3 pre-mRNA cells were treated with 12.5 μM salubrinal for 48 h and SOCS3 expression was induced by treatment with doxycycline with indicated concentrations for 3 h. eIF2α phosphorylation ((p)S51), eIF2α, SOCS3, and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. c PKR expression was eliminated in HEK293 FlpIn SOCS3 pre-mRNA cells using a CRISPR/Cas9 approach (PKR^−/−). SOCS3 expression was induced by 1 μg/mL doxycycline for the times indicated. PKR, SOCS3, and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoforms of SOCS3. Right panel: quantification of relative SOCS3 protein expression of the long and short SOCS3 isoform. Expression of the long isoform was normalized to the expression of the short isoform. Representative results of n = 3 independent experiments are shown. d PKR expression was eliminated in HepG2, Hek293, and HeLa cells using a CRISPR/Cas9 approach. Cells were stimulated with hyIL-6 (50 ng/mL) for 90 min or left untreated as indicated. PKR, SOCS3, (p)Y705 STAT3, STAT3, and tubulin protein expression were evaluated by Western blotting. Arrowheads indicate the long and short isoform of SOCS3. Representative results of n = 3 independent experiments are shown