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. 2009 Nov 11;298(3):C714–C724. doi: 10.1152/ajpcell.00011.2009

Fig. 4.

Fig. 4.

Association of IL-18 with F-actin by fluorescence resonance energy transfer (FRET). I: control (buffer) and TNF-α-treated PMNs were fixed and incubated with primary antibodies to F-actin and IL-18 followed by fluorescently labeled secondary antibodies such that the F-actin immunoreactivity is green, the IL-18 immunoreactivity is red, and the nuclei are blue (bis-benzimide). Negative controls (no primary antibodies) are shown in A and demonstrate that there is no significant cellular fluorescence from incubation with the 2 fluorescently labeled secondary antibodies, and a FRET+ interaction was not observed (F). In B the buffer-treated PMNs demonstrate colocalization of the IL-18 and F-actin immunoreactivity, which also demonstrated a FRET+ interaction between F-actin and IL-18 in control PMNs (G) with a FRET efficiency of 34%. After 3 min of stimulation with TNF-α, the IL-18 immunoreactivity (red) moved to the cell periphery (C), with a decrease in the FRET+ interaction between IL-18 and F-actin (H) with a FRET efficiency of 8%. Moreover, the decrease in IL-18 immunoreactivity continues at 5 and 10 min of TNF-α stimulation (D and E) with a concomitant decrease in FRET-positive pixels (I and J). I, A–J, are representative of 2 identical experiments with different donors and FRET efficiencies of 34 ± 4% between IL-18 and F-actin in control PMNs. II: quantification of the number of FRET+ (IL-18 + F-actin) pixels per cell. *Statistical significance between buffer-treated controls and TNF-α treatments (P < 0.05). The quantification employed 10 separate cells per treatment for each donor. III: further controls that examine the colocalization of phospho-p40phox and Gαi-1 that do not demonstrate a FRET+ interaction. Isolated PMNs were treated for 3 min with TNF-α (10 ng/ml), and the PMNs were fixed, permeabilized, and incubated with primary antibodies to phospho-p40phox (S 315) and the heterotrimeric G protein subunit Gαi-1 followed by incubation with labeled secondary antibodies such that the phospho-p40phox is red (A) and Gαi-1 is green (B). The colocalization (yellow demarcated by white arrows) is seen in C; however, there was not a FRET+ association between these 2 proteins as demonstrated by the absence of color in D, using pseudocolor with arbitrary linear units of fluorescent intensity (a.l.u.f.i.).