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1.
FIGURE 2.

FIGURE 2. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP-mediated anti-apoptotic signaling requires the production of cAMP but does not involve insulin autocrine signaling. A, INS-1 cells were treated without or with STS (100 nm) ± 10 nm GIP for 6 h in the presence or absence of the adenylate cyclase inhibitor, MDL-12,300A (200 μm), and cell death was determined (mean ± S.E. (n = 5); #, p < 0.05 versus DMSO control; $, p < 0.05 versus STS without GIP; %, p < 0.05 versus STS plus GIP without MDL-12,300A). B, INS-1 cells were treated without or with STS (100 nm) plus increasing concentrations of insulin (0–100 nm), IGF-I (10 nm), GIP (10 nm), or forskolin (1 μm) for 6 h (mean ± S.E. (n = 8); #, p < 0.05 versus DMSO control; $, p < 0.05 versus STS without GIP; %, p < 0.05 versus STS plus GIP without MDL-12,300A).

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.
2.
FIGURE 4.

FIGURE 4. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP-mediated anti-apoptotic signaling in STS-treated INS-1 cells does not require transcriptional changes. A and B, INS-1 cells were treated without or with STS (100 nm) ± 10 nm GIP for 4 h (A) or 6 h (B) in the presence or absence of the mRNA translation inhibitor, cycloheximide (CHX, 10 μg/ml), and Western analysis was performed on cell lysates with indicated antibodies (A) or cell death determined (B). In B, mean ± S.E. of cell death (n = 4); #, p < 0.05 versus DMSO control; $, p < 0.05 versus STS without GIP; %, p < 0.05 versus STS plus CHX without GIP.

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.
3.
FIGURE 7.

FIGURE 7. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP-mediated suppression of p38 MAPK and JNK also promotes the survival of INS-1 cells exposed to ER and genotoxic stress. A, INS-1 cells were treated without or with STS (100 nm), thapsigargin (500 nm), or etoposide (5 μm) ± 10 nm GIP for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. Shown are representative blots of at least three independent experiments. Anti-β-actin blot was an internal control. B, INS-1 cells were treated without or with STS, thapsigargin, or etoposide ± GIP for 6 h, and cell death was determined (mean ± S.E. of cell death (n = 6); #, p < 0.05 as indicated). C, INS-1 cells were treated without or with STS, thapsigargin, or etoposide ± p38i (5 μm) or JNKi (5 μm) for 6 h, and cell death was determined (mean ± S.E. of cell death (n = 6); #, p < 0.05 versus STS only; $, p < 0.05 versus thapsigargin only; %, p < 0.05 versus etoposide only).

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.
4.
FIGURE 1.

FIGURE 1. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP inhibits the mitochondria-mediated apoptotic pathway in STS-treated INS-1 cells. A, INS-1 cells were treated without or with STS (100 nm) ± 10 nm GIP for 0–8 h, and onset of apoptosis was determined (mean ± S.E. (n = 4); #, p < 0.05 versus DMSO control; $, p < 0.05 versus STS without GIP). B, INS-1 cells were treated without or with STS plus increasing concentrations of GIP (0–100 nm) for 6 h, and onset of apoptosis was determined (mean ± S.E. (n = 7); $, p < 0.05 versus DMSO control; #, p < 0.05 versus STS without GIP). In the upper right is the concentration-survival response with the calculated EC50 value. C, INS-1 cells were treated without or with STS ± 10 nm GIP for 0–4 h, and Western analysis was performed on mitochondrial or cytoplasmic protein fractions with the indicated antibodies. D, INS-1 cells were treated without or with STS ± 10 nm GIP for 0–6 h, and Western analysis was performed on whole cell lysates with indicated antibodies. Shown in C and D are representative blots of at least three independent experiments. Anti-β-actin and anti-COX IV were internal controls.

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.
5.
FIGURE 6.

FIGURE 6. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP suppresses p38 MAPK and JNK activation via Akt-mediated inhibition of ASK1 in INS-1 cells and human islets. A, INS-1 cells transfected without or with GFP or human ASK1 were treated without or with STS (100 nm) ± 10 nm GIP for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. B, untransfected INS-1 cells were treated without or with STS ± GIP for 4 h, and ASK1 in vitro kinase assays were performed on ASK1 protein that was immunoprecipitated with anti-ASK1 antibody. C, the mean ± S.E. change in ASK1 in vitro kinase activity relative to control is shown (n = 5). D, INS-1 cells transfected with GFP or dominant negative ASK1 (ASK1kinase-dead) were treated without or with STS (100 nm) for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. E, INS-1 cells transfected with wild-type ASK1 (ASK1WT) or ASK1 containing a S83A mutation (ASK1S83A) were treated without or with STS (100 nm) ± 10 nm GIP for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. F, human islets were treated without or with STS (100 nm) ± 10 nm GIP for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. Shown in D and E are representative blots of three independent experiments. Anti-β-actin and anti-GST (GST-Mek 3/6) blots were internal controls. Note: anti-ASK1 antibody detects both human and rat ASK1 protein.

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.
6.
FIGURE 3.

FIGURE 3. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP dynamically regulates mitochondrial levels of bad and bimEL and activation of mitochondrial bax. A, INS-1 cells were treated without or with STS (100 nm) ± 10 nm GIP for 4 h, and Western analysis was performed on mitochondrial and cytoplasmic protein fractions and total cell lysates with indicated antibodies. B–D, for quantification, protein levels were normalized to β-tubulin (cytoplasmic, B), COX IV (mitochondrial, C), or β-actin (cell lysates, D) (mean ± S.E. changes in protein level relative to control; #, p < 0.05; n = 3–6). E, INS-1 cells were treated without or with STS ± 10 nm GIP for 4 h, and Western analysis was performed with the indicated antibodies on mitochondrial protein fractions that were incubated with 10 mm bismaleimidohexane (BMH) for 30 min at room temperature. Shown are representative blots of at least three independent experiments. Anti-COX IV was an internal control. F, INS-1 cells were treated without or with STS plus increasing concentrations of bax channel blocker (0–5 μm) for 6 h, and cell death was determined (mean ± S.E. of cell death (n = 5); #, p < 0.05 versus STS alone).

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.
7.
FIGURE 5.

FIGURE 5. From: Suppression of p38 MAPK and JNK via Akt-mediated Inhibition of Apoptosis Signal-regulating Kinase 1 Constitutes a Core Component of the ?-Cell Pro-survival Effects of Glucose-dependent Insulinotropic Polypeptide.

GIP mediates anti-apoptotic signaling via dual suppression of p38 MAPK and JNK. A, INS-1 cells were treated without or with STS (100 nm) ± 10 nm GIP for 4 h in the absence or presence of Akt inhibitor IV (Akt IV, 400 nm), and Western analysis was performed on total cell lysates with the indicated antibodies. B, INS-1 cells transfected with GFP or dominant negative Akt (Akt-DN) were treated without or with STS (100 nm) ± 10 nm GIP for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. C, INS-1 cells transfected with scramble or Akt 1 and 2 siRNA were treated without or with STS (100 nm) ± 10 nm GIP for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. Of note, total protein levels of Akt were reduced 60–70% in cells transfected with Akt 1 and 2 siRNA. D, INS-1 cells were treated without or with STS ± p38 MAPK inhibitor (p38i, SB 203580, 5 μm) or JNK inhibitor (JNKi, SP 600125, 5 μm) for 4 h, and Western analysis was performed on total cell lysates with the indicated antibodies. E, INS-1 cells were treated without or with STS ± GIP, p38i, JNKi, or p38i and JNKi for 6 h, and cell death was determined (mean ± S.E. of cell death (n = 6); #, p < 0.05 versus STS alone). F, INS-1 cells were treated without or with STS ± p38i and JNKi for 4 h, and Western analysis was performed on mitochondrial and cytoplasmic fractions with the indicated antibodies. G, INS-1 cells were treated without or with STS ± p38i and JNKi for 4 h, and Western analysis was performed on mitochondrial fractions that were incubated with 10 mm bismaleimidohexane (BMH) for 30 min at room temperature. Shown in A–D, F, and G are representative blots of at least three independent experiments. Anti-β-actin and anti-COX IV blots were internal controls.

Scott B. Widenmaier, et al. J Biol Chem. 2009 October 30;284(44):30372-30382.

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