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Notes: Serum-starved, bone marrow-derived macrophages were exposed to apoptotic or necrotic cells, or latex beads (control), and phosphorylation of a variety of signaling molecules was investigated by Western blot. Anti-ACTIVE^® MAPK pAb, Anti-ACTIVE^® p38 pAb, Anti-ACTIVE^® JNK pAb and Anti-ERK 1/2 pAb were used. (3428)

Notes: The authors of this study investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in neural cell apoptosis during retinal development and ischemic injury. Nucleotides 283 to 713 of the ASK1 cDNA were amplified by PCR and cloned into the pGEM^®-T Easy Vector, and sense and antisense probes for in situ hybridization experiments were generated. Anti-ACTIVE^® p38 polyclonal antibody was used for immunohistochemistry analyses to investigate the localization of phosphorylated p38 in mouse retina. (3530)

Notes: Serum-starved PC3 and LNCaP prostate cancer cells were treated with various concentrations of ghrelin over a time course. Western blots of cell lysates were probed with Anti-ACTIVE^® JNK pAb, Anti-ACTIVE^® p38 pAb, Anti-ACTIVE^® MAPK pAb and Anti-ERK(1/2) pAb to assess activation of MAPK pathways. (3434)

Notes: The authors of this study examined the relationship between pathways through which growth hormone (GH), insulin-like growth factor I (IGF-I), and caloric restriction regulate lifespan. Normal and long-lived GH-receptor knockout (GHRKO) mice were compared with transgenic mice overexpressing bovine GH (short lived). When all mice were subjected to caloric restriction, the Normal and GHRKO mice showed decreases in phosphorylation of hepatic Akt, increases in PPAR-gamma and increases in FOXO I transcription. The AntiACTIVE^® p38 pAb was used to examine p38 activity in all three types of mice subjected to caloric restriction. Active p38 was increased in normal and GHRKO mice compared to the transgenic mice and normal mice fed ad lib. The authors show that the Akt/FOXO pathway plays a major role in regulating longevity and provides a link between IGF-1, caloric restriction and growth hormone regulation of lifespan. (3652)

Notes: The authors of this study investigated the role of Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response. RAW264.7 macrophages were transfected with wildtype or mutant SHIP1 or control plasmid. Anti-ACTIVE^® JNK and p38 antibodies were used in Western analyses to determine phosphorylation of these kinases in response to overexpression of SHIP1 or mutant SHIP1. To investigate any effects on IκB-alpha and NF-κB expression, RAW264.7 macrophages were cotransfected with pGL3-XκB-luciferase reporter plasmid and pRL-TK Renilla luciferase control plasmid. Transfected cells were treated with LPS for 6 hours or left untreated (control). The Dual-Luciferase^® Reporter Assay System was used to monitor reporter gene expression. (3524)

Notes: In this report, the innate immune response triggered by Salmonella enterica in Caenorhabditis elegans was studied.  It was found that Salmonella-elicited cell death requires activation of a p38 mitogen-activated protein kinase homolog encoded by the gene pmk-1. Activation of the p38 homolog was monitored by Western Blot using the Anti-ACTIVE™ p38 pAb. Genetic screens in both organisms were undertaken to further understand the interaction of this infection and its response.  (2704)

Notes: The role of integrin alpha(IIb)beta(3) in focal adhesion kinase activation and MAPK signaling was studied using Chinese hamster ovary and human kidney 293 cell lines expressing either the Leu(33) or Pro(33) isoform of beta(3). Compared with Leu(33) cells, Pro(33) cells demonstrated substantially greater activation of ERK2 (but not MAPK family members JNK and p38) upon adhesion to immobilized fibrinogen (but not fibronectin), and upon integrin cross-linking. ERK2 activation was mediated through MAPK kinase and required phosphoinositide 3-kinase signaling and an intact actin cytoskeleton.  Levels of activated MAPK family members ERK1 and 2, JNK, and p38 were assessed by western blotting using Anti-ACTIVE^® MAPK (1:5000 dilution), Anti-ACTIVE^® JNK (1:5000 dilution), and Anti-ACTIVE^® p38 (1:1000 dilution) pAbs.  Anti-ERK 1/2 pAb was used at a 1:5000 dilution as a control for total protein amounts loaded on the blots.  (2789)

Notes: The role of JNK, p38 and MAPK signaling pathways in delayed neuronal death after transient forebrain ischemia was examined using Western blotting and immunohistochemistry. Gerbil hippocampi were homogenized, subjected to SDS-PAGE, blotted to PVDF membranes and incubated with one of the following primary antibodies: Anti-ACTIVE® JNK pAb (1:2000 dilution), Anti-ACTIVE® p38 pAb (1:1000 dilution), and the Anti-ACTIVE® MAPK pAb (1:2000 dilution).The secondary antibody was the Donkey Anti-Rabbit IgG (H+L), AP, diluted 1:10,000 for JNK and p38 and 1:5000 for ERK. For immunohistochemistry, gerbils were perfused with 4% paraformaldehyde. Frozen 40µm sections were stained with either the Anti-ACTIVE® JNK pAb and the Anti-ACTIVE® p38 pAb. (2402)

Notes: The signaling pathways involved in arachidonic acid-stimulated cell adhesion of  human breast carcinoma cells to collagen type IV were examined. MAPK and JNK did not appear to play a role in mediating adhesion but p38 activation appears to be necessary. Levels of activated p38 was determined in MDA-MB-435 cell extracts by Western blot using the Anti-ACTIVE® p38 pAb. Whole cell lysates were subjected to SDS-PAGE, transferred to PVDF membranes, and incubated with the Anti-ACTIVE® p38 (1:1000 dilution). (2405)

Notes: Early steps of embryogenesis are characterized by a series of symmetric and synchronous cell divisions. The authors show that p38 MAPK is asymmetrically activated on one side of early zebrafish embryos.  Dually phosphorylated p38 protein was detected in lysates prepared from one-cell stage embryos by Western blot analysis with the Anti-ACTIVE® p38 pAb. Immunohistochemistry to detect active p38 was also performed on embryos fixed in 4% paraformaldehyde, permeabilized in methanol, and stained with the  Anti-ACTIVE® p38 pAb at a 1:50 dilution. Active p38 levels were also determined by Western blot analysis in 293T cells expressing p38, MKK3, JNK, or human constitutively active form of zebrafish MKK4. (2406)

Notes: Murine 82L fibroblasts were transfected with truncated EGF receptor. In these cells, EGF, PDGF and PMA greatly increased activation of Erk 1 and 2 as judged by Western blotting with the Anti-ACTIVE® p38 pAb, Rabbit, (pTGpY). The treatments also caused the protein caveolin-1 to be phosphorylated as judged by gel shifting and recognition by an anti-tyrosine antibody after immunoprecipitation. The immunoreactivity was reduced when the immunoprecipitates were treated with Calf Intestinal Alkaline Phosphatase. The mobility shift could be reduced by preincubating the cells with either 10 or 50nM of MEK Inhibitor U0126 for 20 minutes prior to a 5-minute treatment with EGF. There was a concomitant reduction in ERK 1/2 phosphorylation as judged by the Anti-ACTIVE® p38 pAb, Rabbit, (pTGpY). (0902)

Notes: The role of JNK and p38 mitogen-activated protein kinase during transient brain ischemia was examined by immunohistochemistry and Western blot analysis. The authors findings indicate that JNK and p38 MAPK pathways may play important roles in neuronal death during brain ischemia. Levels of active JNK and p38 in mouse brain were determined by Western blot analysis and immunohistochemistry using the Anti-ACTIVE® JNK pAb and the Anti-ACTIVE® p38 pAb. For Western blot analysis the antibodies were diluted 1:5000 while immunohistochemical studies were done with dilutions of 1:1000 and 1:400, respectively. Both JNK and p38 were activated following reperfusion of the ischemic tissue. (2409)

Notes: The effect of suppressor of cytokine signaling (SOCS) expression on the Jak/STAT, MAPK, JNK, and p38 signaling pathways was examined in HepG2, Cos-7, and NIH 3T3 cells. Both PMA and bFGF (Promega) resulted in a rapid upregulation of SOCS-3 expression.  MAPK, JNK, and p38 activation was monitored by Western blot analysis using the Anti-ACTIVE® MAPK Anti-ACTIVE® JNK pAb, and the Anti-ACTIVE® p38 pAb. Total levels of active and inactive MAPK protein was determined using the Anti-ERK 1/2 pAb, Rabbit. (2380)

Notes: Mouse embryo fibroblasts from both wildtype and Erk1 knockout mice were stimulated with serum. Erk1 and Erk2 from wildtype mouse embryo fibroblasts were shown to be phosphorylated using the Anti-ACTIVE^® MAPK pAb in a Western blot analysis. In embryo fibroblasts from knockout mice, only Erk2 as phosphorylated. Total Erk1 and Erk2 proteins were detected with the Anti-ERK 1/2 pAb.  (0577)

Notes: Authors studied the effect of chemokines on signaling pathways in primary rat hippocampal neuron cultures. Used Anti-ACTIVE^® MAPK, JNK, and p38 pAbs for western analysis of extracts derived from pure (glial free) hippocampal neuron cultures. (0685)

Notes: The Anti-ACTIVE^® p38 pAb was used for Western blots to follow the activation of p38 in HeLa cells, HL60 cells and human neutrophils following treatment with arachidonic acid. (1043)