Journal: Journal of hepatology

Article Title: Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation

doi: 10.1016/j.jhep.2018.01.036

Figure Lengend Snippet: (A) Gsk3β wild-type (WT) and knockout (KO) bone marrow-derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) for 0 min, 30 min, 1 h, and 6 h. Total cellular proteins were extracted and analysed by western blotting with anti-phosphorylated signal transducer and activator of transcription 1 (p-Stat1), signal transducer and activator of transcription 3 (p-Stat3), signal transducer and activator of transcription 6 (p-Stat6), NF-κBp65 (Ser536) (p-NF-kBp65), cyclic AMP response element-binding protein (Ser133) (p-CREB), AMPK (Thr172) (p-AMPK(T172)), AMPK (Thr479) (p-AMPK(T479)), and acetyl-CoA carboxylase (p-ACC) (Ser79), anti-SHP, anti-Gsk3β, and anti-β-actin. (B) Gsk3β WT (top) and KO BMMs (bottom) were treated with either vehicle or compound C before LPS stimulation. Cellular proteins were harvested after 0, 3, 6, 12, and 24 h of stimulation and analysed by western blotting with anti-SHP or anti-β-actin. (C) Gsk3β WT BMMs were treated with vehicle control or the Gsk3 inhibitor SB216763 before the addition of LPS. Cellular proteins were harvested after 0, 3, 6, and 12 h of stimulation and analysed by western blotting with anti-p AMPK(T172) and anti-SHP or anti-β-actin. (D). Gsk3β WT and KO BMMs were stimulated with LPS (top panel), or Gsk3β WT BMMs were treated with vehicle control or SB216763 before the addition of LPS (middle panel), or Gsk3β KO BMMs were treated with either vehicle or compound C before LPS stimulation (bottom panel). Total cellular RNA was prepared and reverse transcribed. Nr0b2 and Hprt1 gene expression levels were measured by quantitative reverse transcription PCR. Average Nr0b2/Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There was three mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. *p <0.05. Comp. C, compound C; Ctl., control; SB, SB216763.

Article Snippet: Antibodies against total and phosphorylated Gsk3β (Ser9), phosphorylated signal transducer and activator of transcription 1 (Stat1), Stat3, Stat6, phosphorylated NF-κBp65 (Ser536), phosphorylated CREB (Ser133), phosphorylated Akt (Ser473 and Thr308), phosphorylated acetyl-CoA carboxylase (ACC) (Ser79), phosphorylated AMPK (Thr172), and β-actin (Cell Signaling Technology, San Diego, CA, USA), phosphorylated AMPK (Thr479) (ARP American Research Products, Waltham MA, USA), and SHP (NROB2; Abcam, Cambridge, MA, USA) were used for western blot analysis.

Techniques: Knock-Out, Derivative Assay, Western Blot, Binding Assay, Control, Reverse Transcription, Gene Expression

Journal: Journal of hepatology

Article Title: Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation

doi: 10.1016/j.jhep.2018.01.036

Figure Lengend Snippet: (A) Wild-type (WT) bone marrow-derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) for 0, 3, 6, 12, or 24 h. Total cellular proteins were extracted and analysed by western blotting with anti-phosphorylated Akt (Thr308) (p-AKT(T308)), Akt (Ser473) (pAKT(S473)), Gsk3β (Ser9) (p-Gsk3β(S9)), AMPK (Thr479) (p-AMPK(T479)), AMPK (Thr172) (p-AMPK(T172), and acetyl-CoA carboxylase (p-ACC) (Ser79), and β-actin. (B) Phosphatase and tensin homologue (Pten) WT or knockout (KO) BMMs (left panel) or Gsk3β S9A mutant knock-in (KI) BMMs (right panel) were stimulated with LPS for 6 or 24 h. Total cellular RNA was prepared and reverse transcribed. Tnf, Il10, Nr0b2, and Hprt1 gene expression levels were measured by quantitative reverse transcription PCR. Average target gene/Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There were three mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. *p <0.05. Ctl, control.

Article Snippet: Antibodies against total and phosphorylated Gsk3β (Ser9), phosphorylated signal transducer and activator of transcription 1 (Stat1), Stat3, Stat6, phosphorylated NF-κBp65 (Ser536), phosphorylated CREB (Ser133), phosphorylated Akt (Ser473 and Thr308), phosphorylated acetyl-CoA carboxylase (ACC) (Ser79), phosphorylated AMPK (Thr172), and β-actin (Cell Signaling Technology, San Diego, CA, USA), phosphorylated AMPK (Thr479) (ARP American Research Products, Waltham MA, USA), and SHP (NROB2; Abcam, Cambridge, MA, USA) were used for western blot analysis.

Techniques: Derivative Assay, Western Blot, Knock-Out, Mutagenesis, Knock-In, Reverse Transcription, Gene Expression, Control