Hlx overexpression leads to proteasome-dependent degradation of STAT4. (A) Endogenous Hlx levels increased as total STAT4 levels declined. Primary human NK or NK-92 cells were harvested at the indicated time points after IL-12/IL-18 stimulation, and Hlx, STAT4, and β-actin protein levels were determined by immunoblotting. (n = 4 experiments in primary NK, n = 2 experiments in NK-92). (B) Hlx overexpression decreased total and Y693 pSTAT4 levels in NK-92 after IL-12/IL-18 treatment. FACS-purified PINCO- or Hlx-expressing NK-92 cells were harvested at indicated time points after IL-12/IL-18 stimulation. Total STAT4 and β-actin levels were assessed by immunoblotting (n = 4 experiments). (C) Proteasome inhibition rescued loss of STAT4 in NK-92 cells overexpressing Hlx. Cells were preincubated for 1 hour with 20 mM MG-132 (MG), an equal amount of DMSO vehicle, or medium alone (—), followed by IL-12/IL-18 treatment for the indicated times. Total protein was harvested, and total STAT4 and β-actin levels were assessed by immunoblotting (n = 3 experiments). (D) Diminished Y693 pSTAT4 levels in Hlx-overexpressing NK-92 cells despite inhibition of new protein synthesis. PINCO- or Hlx-expressing NK-92 cells were preincubated for 30 minutes with 10 μM CHX, followed by IL-12/IL-18 treatment for the indicated times. Total cellular protein was harvested, and total STAT4 and β-actin levels were assessed by immunoblotting (the arrowhead indicates the position of the 80 kDa marker). (E) Comparison of CHX compared with ethanol (EtOH) carrier effects on STAT4 expression in PINCO- (P) and Hlx-expressing NK-92 after IL-12/IL-18 stimulation for the indicated times (n = 3 experiments).