SOCS-2 abrogates IFNγ-induced GAS luciferase reporter activity. (A) HEK293 cells were transfected with pGL3-8xGAS reporter vector, together with either parental pcDNA vector (w/o SOCS-2) or pcDNA-SOCS-2 expression vector (SOCS-2) for 24 hours. HEK transfectants were then stimulated with indicated concentrations of IFNγ for 16 hours. The luciferase activities were determined by Dual-Luciferase reporter assay system using FusionTM-αFP microplate reader. (B) HEK293 cells were transfected with pGL3-8xGAS reporter vector and either pcDNA (parental) or pcDNA-SOCS-2 expression vector at indicated ratio for 24 hours. Cells were then stimulated with mock (Ctrl) or IFNγ (10 U/mL) for 16 hours. The luciferase activities were determined as described for panel A. All data were plotted as mean values ± SEM of at least 3 independent experiments in duplicates. The statistical analysis was done by comparison between groups using the Student t test. *P < .05 was considered statistically significant. (C) The overexpression of SOCS-2 protein in HEK transfectants was verified by Western blotting using specific anti–SOCS-2 antibodies. Actin was shown as loading control. Vertical line has been inserted to indicate a repositioned gel lane. (D) The role of SOCS-2 in inhibition of STAT1 phosphorylation was verified by overexpression study. HEK cells transiently transfected with pcDNA (lane 3, Ctrl) or pcDNA-SOCS-2 (lane 4, S2) were subjected to IFNγ treatments (20 U/mL) as indicated. Untreated (lane 1, UT) and lipofectamine treated (lane 2, LF) were shown as control. Overexpression of SOCS-2 transcripts was confirmed by RT-PCR (D top panel). Cell lysates were examined by Western blotting using anti-pSTAT1 antibodies, whereas the overexpression of SOCS-2 was verified using anti–SOCS-2 antibodies (D bottom panel). Total STAT1 was probed as a loading control. (E) To further confirm the role of SOCS-2 in inhibition of IFNγ signal transduction, CD14⁺ PBMos were transfected with either control siRNA or SOCS-2 siRNA, and later treated with Tat for 5 hours as indicated. The siRNA-mediated SOCS-2 knockdown in PBMos was verified using RT-PCR (lane 4 vs lane 3). (F) PBMos transfected with either control siRNA or SOCS-2 siRNA were pretreated with Tat for 5 hours and followed by IFNγ treatment for 10 minutes. The phosphorylation levels of STAT1 were examined by Western blotting using anti-pSTAT1 antibodies (lane 4 vs lanes 3 and 5). Total STAT1 was probed as a loading control. The levels of phosphorylation were normalized with total STAT1, whereas the fold changes of individual treatment with reference to the untreated were indicated. Fold change refers to measurement of the indicated band by densitometry. A representative figure of 6 independent experiments on PBMos from different donors was shown. Vertical lines have been inserted to indicate a repositioned gel lane.