FIGURE 6.

AGR2 regulates EGFR and AKT phosphorylation through AREG. A and B, immunoblots of total and phosphorylated AKT in H460 (A) and JH-EsoAd1 (B) cell lysates (8 μg). Cell lysates were derived from control (WT) and AGR2 KD cells. Shown is one representative blot of three independent experiments. The quantified relative densities are shown below the blots and represent the pAKT/total AKT ratio normalized to wild-type cells, which is set at 1.0. C, pAKT/total AKT ratio as determined by protein immunoblotting of H460 cells as noted. Cells were serum starved for 48 h followed by treatment with either 1 μg/ml of anti-AREG IgG or control rabbit IgG for 2 h. The values represent the mean ± 1 S. D. of three independent experiments. Statistical significance between the presence of anti-AREG antibodies and the cell line used (WT, KD, or AREG overexpression) were analyzed by two-way ANOVA (p = 0.0090). D, AREG rescue of AGR2 knockdown. Immunoblots of total and phosphorylated AKT without (−) and with (+) AREG overexpression by cDNA transfection of H460-AGR2KD (left) and JH-EsoAd1-AGR2KD (right) cells. E and F, protein immunoblots of WT and KD H460 (E) and JH-EsoAd1 (F) cell lysates for phosphorylated and total EGFR. Also included are H460-AGR2KD cells in which AREG was overexpressed by transfection (E). The immunoblots were probed with anti-phosphorylated EGFR, total EGFR, and β-actin antibodies. Shown below the immunoblots are graphs depicting the quantified bands normalized to β-actin. The top graphs represent the density of phosphorylated EGFR and total EGFR normalized to β-actin for H460 (G) and JH-EsoAd1 (H) cells. The bottom graphs represent the pEGFR/total EGFR ratio for the same cells.