Journal: Cell Communication and Signaling : CCS

Article Title: MicroRNA let-7b inhibits keratinocyte differentiation by targeting IL-6 mediated ERK signaling in psoriasis

doi: 10.1186/s12964-018-0271-9

Figure Lengend Snippet: let-7b regulated the cell differentiation by reduces p-ERK1/2 signaling in vivo and in vitro. a HaCaT cells were transfected with let-7b mimics or with let-7b inhibitor (“antago let-7b”). Western blotting analysis of phosphorylated ERK1/2 (p-ERK1/2) and total-ERK (ERK1/2). b Immunohistochemistry analysis of p-ERK1/2 analyzed in healthy (n = 4) and psoriasis lesioned skin samples (n = 4). c Western blot analysis shows that p-ERK1/2 is decreased in skin tissue and in primary keratinocytes from keratinocyte-specific let-7b over-expression mice. Expression of total ERK shows no significant difference. Each protein sample results from pools of three mice with the same genotype. d Western blot analysis was performed to detect expression of p-ERK1/2 and total ERK1/2 in transgenic and control mice at days 3 and 5 after treatment with IMQ. e Immunofluorescent staining with p-ERK1/2 ( brown ) antibodies was performed in normal skin and after treatment with IMQ for 3 days in transgenic and control mice. Scale bars, 100 μm. f , g HaCaT cells were treated with MEK-ERK inhibitors (PD98059: 10 μM) and let-7b inhibitor. The differentiation markers was detected by Western blot ( f ) and RT-PCR ( g ). The results obtained analysis were done using t-test. * P < 0.05; ** P < 0.01. (h) Phenotypic presentation of mouse back skin for wild-type treated with IMQ and ERK inhibitor (PD98059) for 7 days. The group treated with DMSO is control group. i Skin thickness was measured on the days indicated in the epidermis of mice treated with IMQ or vehicle as well as added PD98059 or DMSO. Symbols represent mean skin thickness ± s.e.m. for five to six mice per group. j mPASI reflecting the intensity of skin inflammation. Two-tailed unpaired Student’s t-test was performed for statistical analysis. Data are shown as means ± s.e.m. k Light microscopy examination of skin sections stained with H&E, or with keratin K14 antibody. Scale bar: 400 μm

Article Snippet: Mouse skin tissues were embedded in paraffin, 6 μm sections were prepared and stained with K14 antibody (1:1000, Covance), IL-6 antibody (1:250, Cell Signaling Technology) and p-ERK1/2 antibody (1:250, Cell Signaling Technology) analyzed by immunohistochemistry methods.

Techniques: Cell Differentiation, In Vivo, In Vitro, Transfection, Western Blot, Immunohistochemistry, Over Expression, Expressing, Transgenic Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Light Microscopy

Journal: Journal of Neuroinflammation

Article Title: Anti-inflammatory effect of cholera toxin B subunit in experimental stroke

doi: 10.1186/s12974-016-0610-y

Figure Lengend Snippet: Effect of CTB on the mRNA expression of cytokines and transcriptional factors in the ischemic brain after MCAO. a The level of IL-1β mRNA expression in the sham ischemic hemisphere of the CTB-sham group was not significantly different from that of the sham group. CTB dose-dependently downregulated the level of IL-1β mRNA expression in the ischemic brain. b The level of TNF-α mRNA expression in the sham ischemic hemisphere of the CTB-sham group was not significantly different from that of the sham group. CTB dose-dependently downregulated the level of TNF-α mRNA expression in the ischemic brain. c The levels of IL-17 mRNA expression in the sham ischemic hemisphere of the CTB-sham group at 24 and 72 h after ischemia were not significantly different from those of the sham group at 24 and 72 h after ischemia, respectively. CTB dose-dependently downregulated the levels of IL-17 mRNA expression in the ischemic brain both at 24 and 72 h after ischemia. d The levels of IL-10 mRNA expression in the sham ischemic hemisphere of the CTB-sham group at 24 and 72 h after ischemia were not significantly different from those of the sham group at 24 and 72 h after ischemia, respectively. CTB did not affect the levels of IL-10 mRNA expression at 24 h after ischemia, while it dose-dependently upregulated the levels of IL-10 mRNA expression at 72 h after ischemia. Anti-CD25 pretreatment had no impact on the levels of IL-10 mRNA expression at 24 h, while it attenuated the up-regulations of IL-10 mRNA expression at 72 h after ischemia, both in the CTB-L group and CTB-H group. e The levels of TGF-β mRNA expression in the sham ischemic hemisphere of the CTB-sham group at 24 and 72 h after ischemia were not significantly different from those of the sham group at 24 and 72 h after ischemia, respectively. CTB did not affect the levels of TGF-β mRNA expression at 24 h after ischemia, while it dose-dependently upregulated the levels of TGF-β mRNA expression at 72 h after ischemia. Anti-CD25 pretreatment had no impact on the levels of TGF-β mRNA expression at 24 h, while it attenuated the up-regulations of TGF-β mRNA expression at 72 h after ischemia, both in the CTB-L group and CTB-H group. f The level of NF-kB p65 mRNA expression in the sham ischemic hemisphere of the CTB-sham group was not significantly different from that of the sham group. CTB dose-dependently downregulated the level of NF-kB p65 mRNA expression in the ischemic brain. g The level of ERK1 mRNA expression in the sham ischemic hemisphere of the CTB-sham group was not significantly different from that of the sham group. CTB did not affect the level of ERK1 mRNA expression in the ischemic brain. h The level of ERK2 mRNA expression in the sham ischemic hemisphere of the CTB-sham group was not significantly different from that of the sham group. CTB did not affect the level of ERK2 mRNA expression in the ischemic brain. Bars represent mean ± SD ( n = 8); * p < 0.05 vs MCAO group, and # p < 0.05 vs CTB-L group

Article Snippet: As much as 30 μg of protein was separated by SDS/PAGE, transferred 2 h to PVDF membranes, the nonspecific binding of antibodies was blocked with 5 % nonfat dried milk in PBS and then incubated with the primary antibodies at 4 °C overnight: a rabbit anti-rat NF-kB p65 antibody (1:2000, Millipore), a rabbit anti-rat p-ERK1/2 antibody (1:1000, Cell Signaling Technology), a rabbit anti-rat β-actin (1:200, Millipore), and a rabbit anti-rat histone H3 (1:100, Millipore).

Techniques: Expressing

Journal: Journal of Neuroinflammation

Article Title: Anti-inflammatory effect of cholera toxin B subunit in experimental stroke

doi: 10.1186/s12974-016-0610-y

Figure Lengend Snippet: Effect of CTB on expression of cytosolic NF-kB p65, nuclear NF-kB p65, ERK1/2, and p-ERK1/2 in the ischemic brain after MCAO. a CTB dose-dependently downregulated the level of cytosolic NF-kB p65 expression in the ischemic brain. b CTB dose-dependently downregulated the level of nuclear NF-kB p65 expression in the ischemic brain. c CTB did not affect the level of ERK1/2 expression in the ischemic brain, but dose-dependently suppressed ERK1/2 phosphorylation. Bars represent mean ± SD ( n = 8); * p < 0.05 vs MCAO group, and # p < 0.05 vs CTB-L group

Article Snippet: As much as 30 μg of protein was separated by SDS/PAGE, transferred 2 h to PVDF membranes, the nonspecific binding of antibodies was blocked with 5 % nonfat dried milk in PBS and then incubated with the primary antibodies at 4 °C overnight: a rabbit anti-rat NF-kB p65 antibody (1:2000, Millipore), a rabbit anti-rat p-ERK1/2 antibody (1:1000, Cell Signaling Technology), a rabbit anti-rat β-actin (1:200, Millipore), and a rabbit anti-rat histone H3 (1:100, Millipore).

Techniques: Expressing

Journal: Cancer letters

Article Title: Ron receptor overexpression in the murine prostate induces prostate intraepithelial neoplasia

doi: 10.1016/j.canlet.2011.09.021

Figure Lengend Snippet: A, The transgenic lines have increases in prostate pathology over time compared to WT prostates. Of note, the Pb derived lines exhibited only typical pathology at 365 days and only progressed to mPINi stages at the latter time points. The ARR2Pb derived lines exhibited the more severe prostate pathology at earlier time points compared to WT prostates and the 42 and 95 lines with the majority of ARR2Pb lines exhibiting mPINi or adenocarcinoma by the end stage time point. (n=3–5 prostates per group/time point). B and C, Prostate lysates isolated from WT and the transgenic lines at the time points noted were evaluated by Western analysis. Increases in the phosphorylation of ERK1/2 (pERK1/2) per total ERK (B) as well as increases in β-catenin expression (C) were observed in the 95 transgenic line. Samples are shown from two separate mice for each time point. Similar data was obtained for all the transgenic lines. Actin serves as a loading control.

Article Snippet: Membranes were probed with antibodies against Ron (1:400, Santa Cruz Biotechnology, Santa Cruz, CA), phospho(p)-ERK1/2 (1:2,000, Cell Signaling, Boston, MA), total ERK1/2 (1:1,000, Cell Signaling, Boston, MA), β-Catenin (1:1,000, Cell Signaling, Boston, MA), or Actin (1:40,000 a gift from Dr. James Lessard, Cincinnati Children's Hospital Medical Center, Cincinnati, OH) overnight followed by either peroxidase-conjugated Goat Anti-Rabbit or peroxidase-conjugated Donkey Anti-Mouse (Jackson ImmuoResearch Laboratories, West Grove, PA).

Techniques: Transgenic Assay, Derivative Assay, Isolation, Western Blot, Expressing