Fig. 6.
Autocrine TGF-α and IL-1α/β contribute to TNF-induced phosphoprotein signaling regulating hepatocyte apoptosis. Primary rat hepatocytes were isolated, treated, and assayed as described in the Supplementary Experimental Procedures for apoptosis studies. Lysates were collected 0, 0.25, 2, 6, 12, and 24 hours following 5 ng/mL TNF treatment of 50 MOI Adv-infected hepatocytes with mock, 10 µg/mL anti-TGF-α, or 10 µg/mL IL-1ra pretreatments to perturb autocrine ligand activity. Lysates were analyzed using multiplexed phosphoprotein assays for (A) p-Akt, (B) p-ERK1/2, (C) p-JNK, and (D) p-p38. (A,B) IL-1ra inhibition treatments were unchanged from uninhibited treatments and thus not shown. Differences between uninhibited and inhibited phosphoprotein signaling time courses were assessed using two-way ANOVA (p-Akt, anti-TGF-α: P= 0.22; p-ERK1/2, anti-TGF-α: P< 10−4; p-JNK, anti-TGF-α: P< 10−4; p-JNK, IL-1ra: p < 0.01; p-p38, anti-TGF-α: P< 0.0003; p-p38, IL-1ra: P= 0.27). (D) Individual time points that did demonstrate significant differences in both comparisons are labeled (*) if P< 0.05. Data are presented as the mean ± SEM of three biological samples.