Journal: PLoS ONE

Article Title: Iron-Ascorbate-Mediated Lipid Peroxidation Causes Epigenetic Changes in the Antioxidant Defense in Intestinal Epithelial Cells: Impact on Inflammation

doi: 10.1371/journal.pone.0063456

Figure Lengend Snippet: Cells were incubated with Fe/Asc (200 µM/2 mM) and Trolox (0.25 mM) for 6 h at 37°C and/or with 5-AZA (10 µM). The protein expression of Interleukin-6 (IL-6) (A, C) and cyclooxygenase 2 (COX2) (B, D) was determined by western blot as described in Materials and Methods. Results represent the means ± SEM of n = 3 independent experiments. *P<0.05, **P<0.01 vs. controls; # P<0.05, ## P<0.01 vs. Fe/Asc.

Article Snippet: The different primary antibodies were added as follow: 1∶1000 rabbit anti-COX-2 (74 kDa; Novus, Oakville, ON), 1∶5000 goat anti-NF-κB (65 kDa; Santa Cruz Biotechnology, Santa Cruz, CA), 1∶5000 rabbit anti-IκBα (39 kDa; Cell signaling, Beverly, MA) and 1∶500 mouse anti-IL-6 (25 kDa; R&D Systems, Minneapolis, MN).

Techniques: Incubation, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Moderate Physical Activity as a Prevention Method for Knee Osteoarthritis and the Role of Synoviocytes as Biological Key

doi: 10.3390/ijms20030511

Figure Lengend Snippet: ( A – E ): IL-1β immunohistochemistry. ( A ) IL-1β immunolabeling was very weak in the synovium of Group 1; ( B ) IL-1β immunolabeling was very weak in the synovium of Group 2; ( C ) IL-1β immunolabeling was moderate in the synovium of Group 3; ( D ) IL-1β immunolabeling was weak in the synovial membrane of Group 4; ( E ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-1-immunolabeling identified among groups. For details, see the text. ( F – J ): IL-4 immunohistochemistry. ( F ) IL-4 immunolabeling was moderate in the synovium of Group 1; ( G ) IL-4 immunolabeling was strong in the synovium of Group 2; ( H ) IL-4 immunolabeling was weak in the synovium of Group 3; ( I ) IL-4 immunolabeling was strong in the synovial membrane of Group 4; ( J ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-4-immunolabeling identified among groups. For details, see the text. ( K – O ): IL-6 immunohistochemistry. ( K ) IL-6 immunolabeling was moderate in the synovium of Group 1; ( L ) IL-6 immunolabeling was strong in the synovium of Group 2; ( M ) IL-6 immunolabeling was very strong in the synovium of Group 3; ( N ) IL-6 immunolabeling was very strong in the synovial membrane of Group 4; ( O ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-6-immunolabeling identified among groups. For details, see the text. ( P – T ): IL-10 immunohistochemistry. ( P ) IL-10 immunolabeling was moderate in the synovium of Group 1; ( Q ) IL-10 immunolabeling was strong in the synovium of Group 2; ( R ) IL-10 immunolabeling was weak in the synovium of Group 3; ( S ) IL-10 immunolabeling was strong in the synovial membrane of Group 4; and, ( T ) Graph representing the densitometric count (Log2 densitometric count − pixel 2 ) of IL-10-immunolabeling identified among groups. For details, see the text. In inserts are the image analyses by the software in which red color represents immunolabeling. ( A – D , F – I , K – N , P – S ): Objective lens, 20×; scale bars: 50 µm. Results were presented as the mean ± SD. ANOVA was used to evaluate the significance of the results. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: After blocking, the sections were incubated overnight at 4°C with rabbit polyclonal anti-IL-1β (ab9787; Abcam), diluted 1:100 in PBS (Bio-Optica); rabbit polyclonal anti-IL-4 (ab9622; Abcam), diluted 1:100 in PBS (Bio-Optica); goat polyclonal anti-IL-6 antibody (sc-1265; Santa Cruz Biotechnology Inc., Dallas, TX, USA) diluted 1:100 in PBS (Bio-Optica); rabbit polyclonal anti-IL-10 (ab34843; Abcam), diluted 1:100 in PBS (Bio-Optica); mouse monoclonal anti-TNF-α antibody (ab199013; Abcam ) diluted 1:100 in PBS (Bio-Optica); mouse monoclonal anti-MMP-13 antibody (sc-81547; Santa Cruz Biotechnology Inc.) diluted 1:100 in PBS (Bio-Optica); rabbit polyclonal anti-lubricin antibody (ab28484; Abcam), diluted 1:100 in PBS (Bio-Optica).

Techniques: Immunohistochemistry, Immunolabeling, Software

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Time-dependent effects of Ucn on STAT3 phosphorylation in HL-1 cells. ( A ) Representative blots of the time-dependent effects of Ucn [10 nM] using anti-pSTAT3 (Y705 and S727) and total anti-STAT3 polyclonal antibodies. Phosphorylation of STAT3 at both Y705 and S727 sites was significantly enhanced after 15 min. However, while STAT3 phosphorylation at Y705 site further increased at 30, 60, and 120 min, STAT3 phosphorylation at S727 site peaked at 15 min, declined after 30 min, and remained stable after 1 and 2 h. The expression of total STAT3 protein was unaffected by incubation of HL-1 cells with Ucn. ( B ) Corresponding clustered column graph of the Western blot presented in . Data are expressed as% of control. * p<0.05, ** p<0.01. (C, E ) Dose-dependent effects of Ucn (ranging from 10 −11 to 10 −6 M and from 10 −12 to 10 −6 M, respectively) after 15 min of incubation using anti-pSTAT1 (Y701), total anti-STAT1, anti-pSTAT3 (Y705), anti-pSTAT3 (S727), and total anti-STAT3 polyclonal antibodies. GAPDH blot is shown as a loading control. Ucn concentrations as low as 10 −11 M were sufficient to phosphorylate STAT3 at both Y705 and S727 residues. STAT1 (Y701) phosphorylation was unaffected at any Ucn concentration. Likewise, the expression levels of total STAT1 and STAT3 proteins were unchanged. ( D, F ) Corresponding clustered column graphs of the Western blots depicted in Figure 2C and 2D, respectively. Data are expressed as% of control. * p<0.05, ** P<0.01.

Article Snippet: The mouse monoclonal anti-Src (B-12) antibody, the monoclonal anti-P-ERK (E-4) antibody, the rabbit polyclonal anti-ERK1 (C-16) antibody, and rabbit polyclonal anti-IL-6 (M-19) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA).

Techniques: Expressing, Incubation, Western Blot, Concentration Assay

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: JAK2 expression in HL-1 cells and Ucn-induced tyrosine phosphorylation of JAK2. ( A ) Western blot analysis of HL-1 cell lysates with antibodies against all 4 members of the Janus family of tyrosine kinases shows that JAK2 is the highly expressed. ( B, C ) Immunoprecipitation of JAK2 followed by Western blot analysis using an anti-P-tyrosine (pY100) monoclonal Ab and polyclonal anti-JAK2 is shown. In B upper panels , lysates from HL-1 cells, either untreated (CON) or treated with Ucn 10 nM for 30 min, in presence or absence of cycloheximide 100 μg/ml (CHX). In C upper panels lysates from HL-1 cells, either untreated (CON), or treated with Ucn 10 nM (with or without pretreatment with AG490 10 μM), prior to being immunoprecipitated with anti-JAK2. Negative control (Neg Con) was prepared as the control, except for use for immunoprecipitation of the anti-JAK2 antibody. In the lower panels , the same membrane from the upper panel was stripped of antibodies and probed for total JAK2, which served as a loading control. A non-precipitated HL-1 cell lysate (non-stimulated control) was used as a positive control in C left column. See Results section for description.

Article Snippet: The mouse monoclonal anti-Src (B-12) antibody, the monoclonal anti-P-ERK (E-4) antibody, the rabbit polyclonal anti-ERK1 (C-16) antibody, and rabbit polyclonal anti-IL-6 (M-19) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA).

Techniques: Expressing, Western Blot, Immunoprecipitation, Negative Control, Positive Control

Journal: Medical Science Monitor Basic Research

Article Title: Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways

doi: 10.12659/MSMBR.914611

Figure Lengend Snippet: Ucn induced IL-6 release and IL-6-mediated phosphorylation/nuclear translocation of STAT3 (Y705). ( A ) ELISA test showing the temporal release of IL-6 in the culture medium of HL-1 cells treated with Ucn 10 nM for 30 min. IL-6 release started after 1 h, peaked at 12 h, and declined thereafter, although remaining detectable at 24 h. Data are presented as the average of 3 independent experiments. ( B ) A TransAM STAT3 assay was used in HL-1 nuclear extracts to detect and quantify nuclear translocation of p-STAT3. Incubation of HL-1 cells with Ucn (10 nM) increased phosphorylation and nuclear translocation of STAT3 (Y705) in a time-dependent manner. Nuclear extracts from HepG2 cells treated with IL-6 (100 ng/ml) were used as positive control. Use of an anti-IL-6 antibody to neutralize the effects of Ucn-induced IL6 release decreased significantly STAT3 phosphorylation at Y705. * p<0.05, ** p<0.01. ( C ) A representative blot probing pSTAT3 (Y705) in the nuclear extracts from HL-1 cells tested in the TransAM assay is shown. ( D ) Western blot analysis of whole lysates of HL-1 cells treated with Ucn (10 nM) for increasing incubation times (5, 15, and 30 min) confirmed that neutralization of secreted IL-6 (using an anti-IL-6 antibody) was sufficient to block STAT3 phosphorylation at Y705, which peaked at 30 min. Overall expression of total STAT3 was unaffected. * p<0.05, ** p<0.01. ( E ) Corresponding clustered column graph of the Western blot shown in Figure 4D. Data are expressed as% of control. * p<0.05, ** P<0.01.

Article Snippet: The mouse monoclonal anti-Src (B-12) antibody, the monoclonal anti-P-ERK (E-4) antibody, the rabbit polyclonal anti-ERK1 (C-16) antibody, and rabbit polyclonal anti-IL-6 (M-19) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, CA).

Techniques: Translocation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Western Blot, Neutralization, Blocking Assay, Expressing