Tat impairs IFNγ-induced phosphorylation of STAT1. (A) PBMos were untreated (UT), mock treated (M), or treated with indicated concentrations of Tat (T) for 4 hours and followed by the treatment of IFNγ (100 U/mL) for 10 minutes. The tyrosine phosphorylation of STAT1 was probed with specific antibodies. Total STAT1 was probed as 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. Vertical line has been inserted to indicate a repositioned gel lane. (B) PBMos were untreated (UT), treated with mock (M), or treated with 10 nM Tat (T) for 4 hours and followed by the treatment of IFNγ (100 U/mL) at indicated time intervals (minutes) as shown on top of each panel. Cell lysates were examined by Western blotting using anti-pSTAT1 or anti-STAT1 antibodies as indicated. (C,D) To evaluate the effects of Tat on the expression levels of IFNγ receptors, PBMos were treated with mock or Tat (10 nM) at indicated time intervals. Total RNA was extracted and examined for the mRNA expression levels of (C) IFNγR1 and (D) IFNγR2 by QRT-PCR. All data presented were plotted as mean values ± SEM of at least 3 independent experiments in duplicates.