Journal: Frontiers in Pharmacology

Article Title: The Protective Effects of Ivabradine in Preventing Progression from Viral Myocarditis to Dilated Cardiomyopathy

doi: 10.3389/fphar.2016.00408

Figure Lengend Snippet: Expression of p38 MAPK in the myocardial tissues of mice on days 35 and 65. (A) Western blot analysis for phospho-p38 MAPK (P-p38) and total-p38 MAPK (T-p38). (B) Densitometric analysis of relative protein levels for P-p38/ T-p38. ∗∗ P < 0.001, versus CON-35; ∗∗∗ P < 0.001 versus untreated CVMC- 65; ∗ P < 0.001 versus CON-65.

Article Snippet: For the Western blot, proteins were separated on polyacrylamide gels and transferred to a PVDF membrane for detection with various antibodies, including primary monoclonal antibodies for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), phospho-p38 MAPK (P-p38) and total-p38 MAPK (T-p38) (Cell Signaling Technology Corporation, USA) and a polyclonal antibody for collagen I, collagen III (Biorbyt Corporation, USA).

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: DC-SIGN and Toll-like receptor 4 mediate oxidized low-density lipoprotein-induced inflammatory responses in macrophages

doi: 10.1038/s41598-017-03740-7

Figure Lengend Snippet: DC-SIGN participated in the TLR4-NFκB pathway. Negative control (NC) or DC-SIGN siRNA was transfected into macrophages treated or not treated with oxLDL (50 μg/ml) or LPS (62.5 ng/ml) for 60 min. Western blot analysis detected the knockdown efficiency of DC-SIGN and the phosphorylation of p38, JNK, IKKε and NFκB ( A and B ), which was quantified by densitometry in 3 independent experiments and presented as relative units (DC-SIGN/α-tubulin, p38, JNK, IKKε and NFκB phosphorylated protein/total protein). The data are expressed as the mean ± SD from 3 independent tests. * P < 0.05, ** P < 0.01 compared with the macrophages not treated with oxLDL or LPS, ## P < 0.01 compared with the NC in the same group. ( C ) Negative control (NC) or DC-SIGN siRNA was transfected into macrophages treated or not treated with oxLDL (50 μg/ml) or LPS (62.5 ng/ml) for 60 min. Nuclear extracts were then prepared and assayed for p65 activation by EMSA.

Article Snippet: Primary antibodies for DC-SIGN, TLR4, α-tubulin, anti-FLAG, anti-His (Abcam, USA), p65 (t-p65), phosphorylated-p65 (p-p65), IKKε (t- IKKε), phosphorylated-IKKε (p- IKKε), p38 MAPK (t-p38), phosphorylated-p38 MAPK (p-p38), c-Jun N-terminal kinase (t-JNK), and phosphorylated-JNK (p-JNK) were purchased from Cell Signaling Technology (MA, USA).

Techniques: Negative Control, Transfection, Western Blot, Knockdown, Phospho-proteomics, Activation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: β-HB treatment reverses sorafenib resistance by shifting glycolysis-lactate metabolism in HCC.

doi: 10.1016/j.biopha.2023.115293

Figure Lengend Snippet: Fig. 5. β-HB treatment reversed sorafenib resistance and enhanced regorafenib sensitivity by inhibiting the B-raf/MAPK pathway in Huh7-SR and Sk-Hep-1-SR cells. (A) Huh7-SR and SK-Hep-1-SR cells were treated with sorafenib (0 and 10 μM) and β-HB (0, 2.5, and 5 mM) for 48 h, and cell viability was then analyzed using an MTT assay. (B) Huh7-SR and SK-Hep-1-SR cells were treated with regorafenib (0 and 5 μM) and β-HB (0, 2.5, 5, and 10 mM) for 48 h, and cell viability was then analyzed using an MTT assay. (C) Western blotting was conducted to analyze the protein expression of phosphorylated (p)- and total (t)-MAPK-related signaling cascades; α-tubulin was used as an internal control. (D) Western blot images from Fig. 4 C were quantified using ImageJ software. (E) Representative contour plots of apoptosis were detected by flow cytometry stained with Annexin V-FITC and PI. (F) Apoptosis rates were assessed in dose-dependent regorafenib with or without β-HB treatment in Huh7-SR and Sk-Hep-1-SR with Annexin V/PI staining. * p < 0.05; * * p < 0.01; * ** p < 0.001 vs. 0 mM β-HB cells. Data are presented as mean ± SD.

Article Snippet: The following antibodies were used in the experiments: HMGCS2 (1:1000, #ab137043, Abcam, Cambridge, UK), hexokinase I (1:1000, #2024, Cell Signaling, Danvers, MA, USA), hexokinase II (1:1000, #2867, Cell Signaling), phosphofructokinase (1:1000, #8164, Cell Signaling), LDHA (1:1000, #3582, Cell Signaling), PDH (1:1000, #3205, Cell Signaling), IDH (1:1000, #8137, Cell Signaling), phosphorylated (p)- and total (t)-B-raf (1:1000, #2696 and #9433, Cell Signaling), p- and t-MAPK kinase (MEK; 1:1000, #2338 and #9122, Cell Signaling), ERK (1:1000, #9101 and #4695, Cell Signaling), β-catenin (1:1000, #8480, Cell Signaling), ZO-1 (1:1000, #8193, Cell Signaling), Vimentin (1:1000, #5741, Cell Signaling), N-cadherin (1:1000, #13116, Cell Signaling) and antiα-tubulin (1:5000 dilution, #T9026, Sigma-Aldrich).

Techniques: MTT Assay, Western Blot, Expressing, Control, Software, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: ERK1/ATF-2 signaling axis contributes to interleukin-1β-induced MMP-3 expression in dermal fibroblasts

doi: 10.1371/journal.pone.0222869

Figure Lengend Snippet: ( a ) After incubation in the presence or absence of the ATF-2 inhibitor SBI-0087702 (10 μM) for 24 h, the cells were stimulated with 100 pM IL-1β or control for 6 h. At the end of the incubation, MMP-3 mRNA expression was measured. ( b, c ) The cells were exposed to 100 pM IL-1β for indicated time intervals. At the end of the incubation, total (t-) and phosphorylated (p-) ATF-2 were detected by western blotting. For the western blotting, cell lysate (10 μg protein) was applied to each lane. Representative results of p- and t-ATF-2 expression ( b ) and the relative density of p-ATF-2 expression in the cells stimulated with 100 pM IL-1β compared to the results at time point 0 ( c ) are shown. ( d, e ) After incubation in the presence or absence of the ATF-2 inhibitor SBI-0087702 (10 μM) for 24 h, the cells were stimulated with 100 pM IL-1β or control for 15 min. At the end of the incubation, t- and p-ATF-2 were detected by western blotting. Representative results of p- and t-ATF-2 expression ( d ) and the relative density of p-ATF-2 expression are shown ( e ). Values are expressed as the mean ± SE of 3 independent experiments. The F values were 53.31 ( a ), 53.50 ( c ) and 17.62 ( e ). The degrees of freedom were 3 ( a ), 5 ( c ) and 3 ( e ). * P < 0.05, compared with 0 h.

Article Snippet: Rabbit monoclonal antibodies against phospho-ERK1/2 (p-ERK1/2, D13.14.4E), total-ERK1/2 (t-ERK1/2, 137F5), phospho-ATF-2 (p-ATF-2), and total ATF-2 (t-ATF-2, 20F1) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Incubation, Expressing, Western Blot

Journal: PLoS ONE

Article Title: ERK1/ATF-2 signaling axis contributes to interleukin-1β-induced MMP-3 expression in dermal fibroblasts

doi: 10.1371/journal.pone.0222869

Figure Lengend Snippet: ( a ) In the cells transfected with ATF-2 and scrambled siRNAs, t-ATF-2 and β-actin were detected by western blotting. ATF-2 siRNA-transfection, but not scramble siRNA-transfection, decreased the protein expression of ATF-2. β-actin was used as an internal standard. ( b ) Relative density of ATF-2 protein expression in siRNA-transfected cells, compared to that of scrambled siRNA transfected cells, is illustrated. ( c ) After the transfection with ATF-2 and scrambled siRNAs, the cells were incubated with or without 100 pM IL-1β for 6 h. At the end of the incubation, MMP-3 mRNA expression was determined. TBP was used as an internal standard. IL-1β-induced MMP-3 mRNA expression was attenuated in cells transfected with ATF-2 siRNA compared with those transfected with scrambled siRNA. ( d, e ) After the transfection with ATF-2 or scrambled siRNAs, the cells were incubated with or without 100 pM IL-1β for 3 days. The representative images of the cell migration ( d ) and the calculated wound area ( e ) were shown. Results are presented as mean ± SE from 3 independent experiments. Values are expressed as the mean ± SE of 3 independent experiments. The T values was 31.66 ( b ). The F value was 137.16 ( c ) and 142.01 ( e ). The degrees of freedom were 2 ( b ), 3 ( c ) and 3 ( e ). * P < 0.05.

Article Snippet: Rabbit monoclonal antibodies against phospho-ERK1/2 (p-ERK1/2, D13.14.4E), total-ERK1/2 (t-ERK1/2, 137F5), phospho-ATF-2 (p-ATF-2), and total ATF-2 (t-ATF-2, 20F1) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Transfection, Western Blot, Expressing, Incubation, Migration

Journal: PLoS ONE

Article Title: ERK1/ATF-2 signaling axis contributes to interleukin-1β-induced MMP-3 expression in dermal fibroblasts

doi: 10.1371/journal.pone.0222869

Figure Lengend Snippet: ( a, b ) After incubation in the presence or absence of ERK inhibitor FR180204 (25 μM) for 1 h, the cells were treated with 100 pM IL-1β for 15 min. At the end of the incubation, phosphorylated (p-) and total (t-) ATF-2 ( a ) and ERK1/2 ( b ) were detected by western blotting. For the western blotting, cell lysate (10 μg protein) was applied to each lane. Representative results of p- and t-ATF-2 ( a ; upper panel) or p- and t-ERK1/2 expression ( b ; upper panel) and the relative density of p-ATF-2 ( a ; lower panel) and p-ERK1/2 expression ( b ; lower panel) compared to non-treated cells are illustrated. ( c, d ) The cells transfected with ERK1, ERK2 and scrambled siRNAs were incubated in the absence and presence of 100 pM IL-1β for 15 min. Representative results of expression of p- and t-ATF-2 and t-ERK ( c ) and the relative density of p-ATF-2 expression compared to scramble siRNA-transfected cells ( d ) are shown. siRNA-transfection clearly inhibited the IL-1β-induced ATF-2 phosphorylation compared with scramble or ERK2 siRNA-transfection. The IL-1β-induced ATF-2 phosphorylation was also attenuated in ERK1 and 2 double knockdown cells. Values are expressed as the mean ± SE of 3 independent experiments. The F values were 9.16 ( a ), 22.71 ( b ) and 49.48 ( d ). The degrees of freedom were 3 ( a ), 3 ( b ) and 7 ( d ). * P < 0.05.

Article Snippet: Rabbit monoclonal antibodies against phospho-ERK1/2 (p-ERK1/2, D13.14.4E), total-ERK1/2 (t-ERK1/2, 137F5), phospho-ATF-2 (p-ATF-2), and total ATF-2 (t-ATF-2, 20F1) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Incubation, Western Blot, Expressing, Transfection

Journal: PLoS ONE

Article Title: ERK1/ATF-2 signaling axis contributes to interleukin-1β-induced MMP-3 expression in dermal fibroblasts

doi: 10.1371/journal.pone.0222869

Figure Lengend Snippet: In dermal fibroblasts, ERK1 contributes to IL-1β evoked the activation of ERK1/ATF-2 signaling axis, which contributes to cellular migration via MMP-3 expression. ERK2 pathway appears to be necessary for activating other transcription factors in fully MMP-3 expression induced by IL-1β.

Article Snippet: Rabbit monoclonal antibodies against phospho-ERK1/2 (p-ERK1/2, D13.14.4E), total-ERK1/2 (t-ERK1/2, 137F5), phospho-ATF-2 (p-ATF-2), and total ATF-2 (t-ATF-2, 20F1) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan).

Techniques: Activation Assay, Migration, Expressing