Journal: Frontiers in Cell and Developmental Biology
Article Title: Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways
Figure Lengend Snippet: Giardia duodenalis trophozoites activated the p38 and ERK/MAPK signaling pathways via TLR9. (A) A total of 3 × 10 6 wild-type (WT) mouse peritoneal macrophages were stimulated with 1 × 10 6 G . duodenalis trophozoites for various periods (0–6 h) following which the phosphorylation levels of p38 and ERK were analyzed by Western blot. (B) The secretion levels of IL-6, TNF-α, and IL-12 p40 in cell culture supernatants were measured by ELISA. (C) Macrophages treated or not with small-interfering RNA (siRNA) targeting TLR9 (siTLR9) were stimulated with Giardia lamblia virus (GLV)–free or GLV-containing Giardia trophozoites for 3 h. (D) A total of 3 × 10 6 WT macrophages pretreated or not with siTLR9 were incubated for 3 h with 1 × 10 6 GLV-free Giardia trophozoites or 1 × 10 6 GLV-containing Giardia trophozoites, following which TLR9 expression levels were analyzed by Western blot. Relative protein expression was quantified by densitometric analysis using β-actin as an internal reference. Data are expressed as means ± SD from three separate experiments. ns, no significant difference, * p
Article Snippet: After blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline 0.1 Tween 20 (TBST) for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies targeting TLR9, p38, ERK, AKT, NF-κB p65, phospho-p38, phospho-ERK, phospho-AKT, phospho-p65, phospho-IκBα, β-actin (all rabbit), and IκBα (mouse) (all from Cell Signaling Technology, Danvers, MA, United States) diluted 1/1,000 in 5% BSA.
Techniques: Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Small Interfering RNA, Incubation, Expressing