Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 1. JNK1 directly binds to TAp63c at its transactivation domain. (A), Hela cells transfected with Flag-JNK1 were lysed and subjected to GST pull-down assay with GST alone or GST-TAD. 10 lg of total protein from the same cell lysates were directly loaded as an input control. The adsorbates were analyzed by immunoblotting analysis (IB) using anti-Flag (top panel). Comparable amounts of GST and GST-TAD fusion proteins were shown by staining the membrane with Coomassie blue R-250 (bottom panel). (B and C) H1299 cells were co-transfected with HA-TAp63c and Flag-JNK1. Whole-cell lysates were subjected to immunoprecipitation (IP) with normal mouse IgG, anti-HA or anti-Flag. IP products were subjected to IB analysis with indicated primary antibodies and trueblot secondary antibody.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Transfection, Pull Down Assay, Control, Western Blot, Staining, Membrane, Immunoprecipitation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 2. Overexpression of JNK1 inhibits TAp63c transactivity. (A) H1299 cells were transfected with a mixture of Bax-Luc and TK-Renilla plus HA-TAp63c or (and) Flag-JNK1 plasmid; (B) H1299 cells were transfected with a mixture of Bax-Luc and TK-Renilla plus HA-TAp63c and increasing amounts of Flag-JNK1 plasmid. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as percentage of Bax-Luc expression with standard deviation (n = 3). Bax-Luc expression in cells transfected with TAp63c alone with Bax-Luc/TK-Renilla mixture was set as 100%. IB analysis was performed to detect the expression of TAp63c and JNK1 using anti-HA and anti-Flag respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Over Expression, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Standard Deviation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 3. Ser12 in TAp63c is critical for JNK1-mediated trans-inhibition. H1299 cells were transfected with Bax-Luc/TK-Renilla mixture plus HA-TAp63c (wild-type or S12A mutant) or (and) Flag-JNK1 plasmid. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as percentage of Bax-Luc expression with standard deviation (n = 3). Bax-Luc expression in cells transfected with wild-type TAp63c alone with Bax-Luc/ TK-Renilla mixture was set as 100 percent. IB analysis was performed to detect the expression of TAp63c and JNK1 using anti-HA and anti-Flag respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Inhibition, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Standard Deviation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 4. Inhibition or knockdown of JNK1 increases TAp63c transactivity. (A) H1299 cells were transfected with Bax-Luc/TK-Renilla mixture with or without HA-TAp63c, and treated with JNK inhibitor SP600125 (JNKin); (B) H1299 cells were transfected with Bax-Luc/TK-Renilla mixture plus HA-TAp63c or (and) siJNK1. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as percentage of Bax-Luc expression with standard deviation (n = 3). Bax-Luc expression in cells transfected with TAp63c alone with Bax-Luc/TK-Renilla mixture was set as 100%. IB analysis was performed to detect the expression of TAp63c, total or phosphorylated (p-JNK1) JNK1 using anti-HA, antibody specific to total or T183-phosphorylated JNK1, respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Inhibition, Knockdown, Transfection, Luciferase, Activity Assay, Expressing, Standard Deviation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 5. JNK1 inhibits TAp63c-mediated cell apoptosis. H1299 cells were transfected with HA-TAp63c and (or) Flag-JNK1. 48 h post transfection, cells were trypsinized and subjected to IB (A) or flow cytometry analysis (B) of apoptosis. Cleaved PARP1 (CL-PARP), JNK1 and TAp63c were detected with antibody to N-terminal cleaved PARP1, Flag, or HA respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Transfection, Cytometry

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