Fig. 3
PTPN14 promotes SOCS7 degradation through ubiquitination at K11 and K48. a, b qRT-PCR (a) and western blotting (b) were used to detect SOCS7 mRNA levels in livers of wild-type and PTPN14-deficient mice stimulated with 0.01 mg/kg LPS and 800 mg/kg D-GalN. c Hep G2 cells were treated with E64d+PepA (lysosomal degradation inhibitor) or MG132 (proteasome degradation inhibitor), stimulated with 1 μg/mL LPS and 5 mM D-GalN for 24 h, and then the interaction between SOCS7 and ubiquitin or PTPN14 were detected by Co-IP. d HA-labeled PTPN14 and Myc-labeled SOCS7 were transfected into Hep G2 cells. Cells were stimulated with 1 μg/mL LPS and 5 mM D-GalN for 24 h, and then the interaction between SOCS7 and ubiquitin was detected by Co-IP. e HA-labeled PTPN14 and Myc-labeled SOCS7 were transfected into wild-type and PTPN14-deficient BMDMs. After stimulation with 1 μg/mL LPS and 5 mM D-GalN for 24 h, the interaction between SOCS7 and ubiquitin was detected by Co-IP. f Schematic diagram of the ubiquitin mutants. g RAW264.7 cells were transfected with different ubiquitin mutants, HA-labeled PTPN14, and Myc-labeled SOCS7, followed by stimulation with 1 μg/mL LPS and 5 mM D-GalN for 24 h. The interaction between SOCS7 and ubiquitin was detected by Co-IP. In d, e, and g, MG132 was added during cell culture to inhibit the degradation of SOCS7. qRT-PCR data (a) were representative of one experiment with at least three independent biological replicates; a single data point represented one technical repeat. Two-tailed Student’s t-test was used to compare the means between the two groups. Blots (b–e, g) were representative of three independent experiments. n.s not significant.
