Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP induces cytokine expression through MAPK. ( A ) J774A.1 macrophages were incubated for 0-60 min with or without ACP (100 µg/mL). The phosphorylation levels of ERK1/2, JNK1/2 and p38 were assayed by Western blot. ( B, C ) J774A.1 macrophages stably transfected with a control shRNA plasmid (sh-SC), a ERK1 shRNA plasmid (sh-ERK1), a JNK1 shRNA plasmid (sh-TLR2), a p38α shRNA plasmid (sh-p38α), and a p38β shRNA plasmid (sh-p38β) were incubated for 24 h with or without ACP (100 μg/mL). The levels of TNF-α ( B ) and IL-6 ( C ) in the culture medium were measured by ELISA. The Western blot results are representative of those obtained in three different experiments. The ELISA data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Expressing, Incubation, Western Blot, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP pretreatment induces LPS tolerance. ( A, B ) J774A.1 macrophages ( A ) and mouse peritoneal macrophages ( B ) were incubated for 24 h with or without ACP (100 μg/mL) or LPS (0.1 μg/mL)(1 st stimulus). After washing, cell cultures were changed for fresh medium and then were incubated for 24 h with or without LPS (1 μg/mL)(2 nd stimulus). The levels of TNF-α and IL-6 in the culture medium were measured by ELISA. ( C ) J-Blue cells were incubated for 24 h with or without ACP (100 μg/mL) or LPS (0.1 μg/mL)(1 st stimulus). After washing, cell cultures were changed for fresh medium and then were incubated for 24 h with or without LPS (1 μg/mL)(2 nd stimulus). The activation levels of NF-κB were measured by an NF-κB reporter assay. (D) J774A.1 macrophages were incubated for 24 h with or without ACP (100 μg/mL). After washing, cell cultures were changed for fresh medium and then were incubated for 0-30 min with or without LPS (1 µg/mL). The phosphorylation levels of ERK1/2, JNK1/2 and p38 were assayed by Western blot. The Western blot results are representative of those obtained in three different experiments. The data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001, compared to 1 st P/2 nd L group. P: PBS; L: LPS.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Reporter Assay, Western Blot