Skip to main content
. 2018 Nov 20;9:2676. doi: 10.3389/fimmu.2018.02676

Figure 3.

Figure 3

MAPKs are required for IVIg-induced IL-10 production in response to LPS. (A) Monocytes from healthy control participants were unstimulated or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or both, for 0, 10, 40, or 120 min. Cell lysates (2.5 × 105 cells / time point) were prepared at the indicated times. Lysates were separated by SDS-PAGE and analyzed by western blotting using phosphospecific antibodies for ERK1/2 and p38, and GAPDH, as a loading control. Results are representative of n = 5 experiments; monocytes were derived from 1 participant for each of 5 independent experiments. Densitometry for pERK1/2 and pp38; normalized to GAPDH and relative to LPS 10 min; are averaged and shown below each band and values are graphed and reported as mean ± SEM. In (B) and (C), monocytes were pre-treated for 1 h with an appropriate volume of DMSO, as a vehicle control, or (B) the ERK1/2 inhibitors, PD98059 (PD) and SCH772984 (SCH), or (C) the p38 inhibitors, SB203580 (SB), and BIRB796 (BIR). In (D) and (E) monocytes were untreated (UnRx) or pre-treated for 48 h with a non-silencing siRNA (ns) or 2 different sets of siRNAs (si1 or si2) to ERK1 and 2 (D) or p38α, p38γ, and p38δ (E). Samples in (D) and (E) were prepared, as above, and analyzed by western blotting using antibodies for ERK1/2 (D) or p38 (E) and GAPDH, as a loading control. Densitometry for ERK1/2 (D) or p38 (E) normalized to GAPDH and relative to untreated control (UnRx) are shown below each band. Monocytes in (B–E) were unstimulated (C) or stimulated with LPS (100 ng/ml), IVIg (5 mg/ml), or (IVIg + LPS) for 24 h. Clarified cell supernatants were assayed for IL-10 by ELISA. Values are reported as mean ± SEM for n = 7 (B,C) or n = 5 or 6 (D,E) participants performed as independent experiments, assayed in duplicate. *p < 0.05, **p < 0.01, and ns = not statistically different for the comparisons indicated. Statistical analyses were performed using a two-way ANOVA in (A) and repeated measures one-way ANOVA in (B–E) with Dunn's multiple comparisons correction.