Figure 6
From: Soy isoflavones and their metabolites modulate cytokine-induced natural killer cell function
Genistein modulates cellular signaling events. PBMC from healthy donors were cultured with 25 µM of genistein or DMSO (vehicle control) for 4 hours then stimulated with 20 ng/ml IL-12 and 50 ng/ml IL-18 for either 24 hours of additional incubation (long-term), or 15 minutes of additional incubation (short-term) to analyze canonical signal transduction events. (a) Immunoblot analysis of pERK was conducted in short-term lysates to focus on signaling events proximal to initial IL-18 receptor engagement. (b) Densitometry analysis of western blot pERK levels across conditions normalized to total ERK and β-actin levels. (c) Immunoblot analysis of pSTAT1 was conducted in long-term lysates where IFN-γ, an upstream STAT1 activating stimulus was at high levels (d) Densitometry analysis of western blot phosphor-STAT1 levels across conditions normalized to total STAT1 and β-actin levels. (e) Immunoblot analysis of pTyr was conducted in long-term lysates where IFN-γ and pan-pTyr activation was at high levels. Data are presented from three separate donors with similar results. In two of three experiments, total STAT1 or ERK, along with β-actin are included as controls and were run using identical lysates on separate gels, given similar molecular weight of some proteins. In one of three experiments, each phospho-specific Ab blot was stripped and re-probed for detection of loading controls. (Veh = vehicle; 12/18 = IL = 12 + IL-18 stimulation) Immunoblots were cropped for presentation purposes and original blots are provided as Supplemental Fig. 4.
