Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular signaling pathways control mitochondrial events associated with the development of ischemia/ reperfusion-associated damage.

doi: 10.1111/j.1432-2277.2009.00883.x

Figure Lengend Snippet: Figure 1 Alterations in MAPK activity are associated with IR or HR. Representative immunoblots (a, c, e) and a summary graph (b, d) are shown. (a, b). Three animals undergoing heterotopic heart transplantation were analysed in each group. Treatment conditions were as follows: Co, untreated animals; H, 45 min of ischemia; R1-R4, hearts exposed to 45 min of ischemia and either 10 min (R1), 2 h (R2), 12 h (R3), 24 h (R4) or 48 h (R5), of reperfusion. BALB/c mouse heart lysates display low levels of ERK1,2 activity in the control group (Co). 45 min of ischemia increased stress kinase activity (p38: 4.6-fold increase, *P < 0.05 vs. control; JNK: 9.4-fold increase, *P < 0.05 vs. control) but left ERK 1,2 unaf- fected. 10 min of reperfusion (R1) caused a significant increase in ERK activity (ERK1,2: 8.4-fold increase, **P < 0.01 vs. control), and a further increase in p38 (p38: 9.4-fold increase, **P < 0.01 vs. control) and JNK (JNK1,2: 57.7-fold increase, **P < 0.01 vs. control) activity. After 2 h of reperfusion (R2) JNK (JNK1,2: 2.6-fold increase) and p38 (p38: 0.6-fold) activity had returned to control (Co) levels, while ERK (ERK1,2: 4.8-fold increase) activation still did not return to prereperfusion levels and stayed above levels in control hearts until the end of the observation period (ERK1,2: 2.73-fold increase). Data are expressed as mean ± SEM (of n = 3). (c–e) Changes in MAPK signaling in HL-1 cells and primary BALB/c car- diomyocytes subjected to 45 min of hypoxia (0.5% O2, 37 C, serum-/glucose-free medium) and up to 48 h of reoxygenation. The treatment conditions were as follows: Co, untreated cells; H, 45 min of hypoxia R1-R4: hypoxia (45 min) followed by reoxygenation in growth medium for 10 min (R1), 2 h (R2), 24 h (R3) or 48 h (R4), respectively. 45 min of hypoxia increased activity of all three MAPKs, 10 min of reperfusion lead to a further increase in their activities. After 2 h of reperfusion JNK and p38 activity had ceased while ERK activity still had not returned to control levels. Data are expressed as mean ± SEM (n = 3, all **P < 0.01). In the case of primary cardiomyocytes a single experiment was performed.

Article Snippet: Immunoblots were probed with the following antibodies: phospho-ERK (sc-7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho JNK (AF-1205; R&D Systems, Minneapolis, MN, USA) or phospho-p38 (9211S, Cell Signaling, Danvers, MA, USA).

Techniques: Activity Assay, Western Blot, Transplantation Assay, Control, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Somatostatin, Cortistatin and Their Receptors Exert Antitumor Actions in Androgen-Independent Prostate Cancer Cells: Critical Role of Endogenous Cortistatin

doi: 10.3390/ijms232113003

Figure Lengend Snippet: Molecular consequences of somatostatin (SST) or cortistatin (CORT) treatment in androgen-independent (AI) prostate cancer (PCa) cells. ( A , B ) Phosphorylation levels of protein belonging to different oncogenic signaling pathways (AKT, ERK, JNK, PTEN and AR) in response to SST and CORT treatment in AI-PCa cells. Phospho-protein levels were normalized by the total amount of each respective protein. Protein data were represented as percent of vehicle-treated cells (set at 100%). ( C ) Fold change in markers of proliferation, migration, and PCa-aggressiveness in response to SST and CORT treatment in AI-PCa cells. Gene expression was represented as the percentage of vehicle-treated cells (set at 100%). Asterisks (* p < 0.05; ** p < 0.01 and *** p < 0.001) indicate statistically significant differences between treatment and vehicle-treated cells. N.D: Non-detected. Ctrl: Control.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween-20 and incubated overnight with the specific primary antibodies at 1:1000 dilution [phospho-AKT (p-AKT; #4060S; Cell-Signaling, Barcelona, Spain), AKT (#9272S; Cell-Signaling, Barcelona, Spain), phospho-ERK (#4370S; Cell-Signaling), ERK (#9102S; Cell-Signaling), phospho-JNK (#AF1206; RD system), JNK (#AF1387; RD system), phospho-PTEN (#S380; Cell-Signaling), PTEN (9552S; Cell-Signaling), phospho-AR (#16969; Cell-Signaling), AR (ab133273; Abcam)].

Techniques: Phospho-proteomics, Protein-Protein interactions, Migration, Gene Expression, Control

Journal: PLoS ONE

Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

doi: 10.1371/journal.pone.0157860

Figure Lengend Snippet: (A) Western blot analysis of endogenous JNK1 present in HEK293T WCE and in NEIL1-FLAG immunoprecipitates. (B) Far-Western analysis to confirm the interaction between NEIL1 and JNK1. Top panel, Coomassie stained SDS-PAGE gel of NEIL1 and its phosphomimetic and ablating mutants (50 pmoles). Bottom panel, membrane after transfer, denaturation, slow refolding, binding to JNK1 from HEK293 WCE, and probing with an anti-JNK1 antibody. BSA was used as a negative control.

Article Snippet: An anti-mouse JNK1 purified monoclonal antibody (for far-Western analysis, clone 228601 from R&D Systems, lot # UQL0209081; cat # MAB17761) and a rabbit polyclonal NEIL1 antibody (for Western Blotting of IP reactions, Abcam, ab21337, lot # GR133490-1) were diluted 1:1000 (final concentration 1μg/ml) in blocking reagent.

Techniques: Western Blot, Staining, SDS Page, Membrane, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

doi: 10.1371/journal.pone.0157860

Figure Lengend Snippet: (A) In vitro kinase assays were performed with NEIL1 constructs expressed from E. coli cells and purified to homogeneity and active JNK1 kinase for 30 minutes at 32°C. γ- 32 P incorporation was quantified using phosphor-autoradiography after SDS-PAGE analysis of the samples. Lane 1, no JNK1 control; lane 2, no NEIL1 control; lane 3–13 are WT, S207E, S207A, S306E, S306A, S61E, S61A, DE, DA, TE, TA, respectively. (B) Graphical representation of two experimental repeats of the in vitro kinase assay. Statistically significant values (at 95% confidence) were determined by a one-way Anova test where * denotes p-values <0.05, *** denotes p-values <0.0005, and **** denotes p values <0.0001. (C) Glycosylase activity assays were performed using Sp:C and AP:C substrates with increasing amounts of in vitro phosphorylated NEIL1 (pNEIL1), unphosphorylated NEIL1 (positive control), and JNK1 (negative control). S and P indicate substrate and product, respectively.

Article Snippet: An anti-mouse JNK1 purified monoclonal antibody (for far-Western analysis, clone 228601 from R&D Systems, lot # UQL0209081; cat # MAB17761) and a rabbit polyclonal NEIL1 antibody (for Western Blotting of IP reactions, Abcam, ab21337, lot # GR133490-1) were diluted 1:1000 (final concentration 1μg/ml) in blocking reagent.

Techniques: In Vitro, Construct, Purification, Autoradiography, SDS Page, Control, Kinase Assay, Activity Assay, Positive Control, Negative Control