Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-AMPK-alpha, phospho (Thr172) Monoclonal Antibody, Unconjugated, Clone 40H9 , Cell Signaling , Cat#2535; RRID: AB_331250.

Techniques: Produced, FLAG-tag, Recombinant, Transfection, Silver Staining, Isolation, Cell Culture, Expressing, Plasmid Preparation, Luciferase, shRNA, Software

Journal: iScience

Article Title: A pref-1-controlled non-inflammatory mechanism of insulin resistance

doi: 10.1016/j.isci.2023.106923

Figure Lengend Snippet:

Article Snippet: Rabbit-anti-P-AMPK Alpha (Thr172) , Cell Signaling Technology , 2535S.

Techniques: Recombinant, Protease Inhibitor, Western Blot, SYBR Green Assay, Transfection, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Immunoprecipitation, Negative Control, Software, Purification

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: LMNA deletion inhibits de novo fat synthesis through AMPKα phosphorylation. ( A ) The expression levels of AMPKα and its phosphorylated form in LMNA -KO 7701 cells are shown with the corresponding plot of the quantitative analysis ( n = 3) ( B ). ( C ) CO-/C IP assays were performed to assess the endogenous interaction between pLamin Aand pAMPKα-T172 or between lamin A/C and pAMPKα-T172 in 7701 cells. IgG was used as a negative control, and the input was used to examine the levels of pAMPKα-T172 and lamin A/C. ( D ) An exogenous reverse pull-down experiment was performed in 7701 cell lines. 7701 cells were transiently transfected with the AMPK α-FLAG plasmid or control vector for 48 h. An anti-FLAG antibody was used to immunoprecipitate the cell lysates. ( E ) HEK293T cells were transiently cotransfected with the plasmid containing the LMNA cDNA and the AMPKα -N, -C and -F plasmid or control vector. An anti-FLAG antibody was used to immunoprecipitate the nuclear lysates. Data are presented as the means ± SD. ns , p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Protein fragments were transferred onto a nitrocellulose (NC) membrane and subjected to immunoblot analyses with the indicated primary antibodies at the following dilutions: anti-lamin A/C (10298-1-AP, Proteintech, Wuhan, China) at 1:1000, anti-phospho-lamin A/C (S22) (2026, CST) at 1:1000, anti-phospho-lamin A/C (S392) (ab58528, Abcam) at 1:1000, anti-AMPK alpha 1 (5831, CST) at 1:1000, anti-phospho-AMPK alpha (T172) (2535S, CST) at 1:1000, anti-acetyl-CoA carboxylase (21923-1-AP, Proteintech) at 1:1000, anti-phospho-acetyl-CoA carboxylase (S79) (3661S, CST) at 1:1000, anti-fatty acid synthase (FASN) rabbit polyclonal antibody (10624-2-AP, Proteintech) at 1:1000, anti– mitochondrial pyruvate carrier (MPC1; 14462, CST) at 1:1000, anti-β-hydroxy-β-methylglutaryl coenzyme A reductase (HMGCR) polyclonal antibody (13533-1-AP, Proteintech) at 1:1000, anti-GAPDH (60004-I-Ig, Proteintech) at 1:5000, anti-β-actin (60008-1-Ig, Proteintech) at 1:5000, and anti-FLAG (80010-1-RR, Proteintech) at 1:5000, which was used as a control.

Techniques: Expressing, Negative Control, Transfection, Plasmid Preparation

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: Abnormalities in Lamin A/C function trigger AMPKα activation. ( A ) Mutations in the LMNA structure and sequence are shown: D203N is located in the rod domain and R482W, G465D, and T528R are located in the LTD. Western blotting was performed to detect changes in lamin A/C, pLamin A/C, AMPKα, and pAMPKα-T172 levels upon the rescue of lamin A/C expression via the transfection of the LMNC cDNA, LMNA-GFP cDNA and LMNA mutants, and the corresponding semiquantitative data obtained after GAPDH normalization are shown. ( B ) TG and lactate synthesis were quantified in the different transfected groups ( n = 3). ( C ) Western blotting was performed to assess concentration-dependent changes in lamin A/C, pLamin A/C, AMPKα, pAMPKα, pACC1 and ACC1 levels in 7701, HepG2 and MHCC97-H cells treated with lonafarnib. The corresponding quantitative data were obtained by standardization with GAPDH. Data are presented as the means of at least three independent experiments ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Protein fragments were transferred onto a nitrocellulose (NC) membrane and subjected to immunoblot analyses with the indicated primary antibodies at the following dilutions: anti-lamin A/C (10298-1-AP, Proteintech, Wuhan, China) at 1:1000, anti-phospho-lamin A/C (S22) (2026, CST) at 1:1000, anti-phospho-lamin A/C (S392) (ab58528, Abcam) at 1:1000, anti-AMPK alpha 1 (5831, CST) at 1:1000, anti-phospho-AMPK alpha (T172) (2535S, CST) at 1:1000, anti-acetyl-CoA carboxylase (21923-1-AP, Proteintech) at 1:1000, anti-phospho-acetyl-CoA carboxylase (S79) (3661S, CST) at 1:1000, anti-fatty acid synthase (FASN) rabbit polyclonal antibody (10624-2-AP, Proteintech) at 1:1000, anti– mitochondrial pyruvate carrier (MPC1; 14462, CST) at 1:1000, anti-β-hydroxy-β-methylglutaryl coenzyme A reductase (HMGCR) polyclonal antibody (13533-1-AP, Proteintech) at 1:1000, anti-GAPDH (60004-I-Ig, Proteintech) at 1:5000, anti-β-actin (60008-1-Ig, Proteintech) at 1:5000, and anti-FLAG (80010-1-RR, Proteintech) at 1:5000, which was used as a control.

Techniques: Activation Assay, Sequencing, Western Blot, Expressing, Transfection, Concentration Assay

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: pLamin A/C is an indicator of increased lipid synthesis. ( A ) Treatment with 25 mM glucose for 12 h promoted lamin A/C phosphorylation and FASN expression in 7701, HepG2 and MHCC97-H cells. Semiquantitative analyses of blots are displayed. ( B ) TG synthesis was quantified ( n = 3). ( C ) Lamin A/C phosphorylation was increased by 25 mM glucose in a time-dependent manner in 7701 and HepG2 cells. Gray values from blots are displayed. ( D ) The inhibition of AMPKα activation was shown in 7701, HepG2 or MHCC97-H cells treated with 25 mM glucose. Semiquantitative analyses of blots are displayed. ( E ) Person’s correlation analysis between pLamin A and FASN or pAMPKα-T172 is shown. Data are shown as the means of at least three independent experiments ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: Protein fragments were transferred onto a nitrocellulose (NC) membrane and subjected to immunoblot analyses with the indicated primary antibodies at the following dilutions: anti-lamin A/C (10298-1-AP, Proteintech, Wuhan, China) at 1:1000, anti-phospho-lamin A/C (S22) (2026, CST) at 1:1000, anti-phospho-lamin A/C (S392) (ab58528, Abcam) at 1:1000, anti-AMPK alpha 1 (5831, CST) at 1:1000, anti-phospho-AMPK alpha (T172) (2535S, CST) at 1:1000, anti-acetyl-CoA carboxylase (21923-1-AP, Proteintech) at 1:1000, anti-phospho-acetyl-CoA carboxylase (S79) (3661S, CST) at 1:1000, anti-fatty acid synthase (FASN) rabbit polyclonal antibody (10624-2-AP, Proteintech) at 1:1000, anti– mitochondrial pyruvate carrier (MPC1; 14462, CST) at 1:1000, anti-β-hydroxy-β-methylglutaryl coenzyme A reductase (HMGCR) polyclonal antibody (13533-1-AP, Proteintech) at 1:1000, anti-GAPDH (60004-I-Ig, Proteintech) at 1:5000, anti-β-actin (60008-1-Ig, Proteintech) at 1:5000, and anti-FLAG (80010-1-RR, Proteintech) at 1:5000, which was used as a control.

Techniques: Expressing, Inhibition, Activation Assay

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: LMNA deletion inhibits de novo fat synthesis through AMPKα phosphorylation. ( A ) The expression levels of AMPKα and its phosphorylated form in LMNA -KO 7701 cells are shown with the corresponding plot of the quantitative analysis ( n = 3) ( B ). ( C ) CO-/C IP assays were performed to assess the endogenous interaction between pLamin Aand pAMPKα-T172 or between lamin A/C and pAMPKα-T172 in 7701 cells. IgG was used as a negative control, and the input was used to examine the levels of pAMPKα-T172 and lamin A/C. ( D ) An exogenous reverse pull-down experiment was performed in 7701 cell lines. 7701 cells were transiently transfected with the AMPK α-FLAG plasmid or control vector for 48 h. An anti-FLAG antibody was used to immunoprecipitate the cell lysates. ( E ) HEK293T cells were transiently cotransfected with the plasmid containing the LMNA cDNA and the AMPKα -N, -C and -F plasmid or control vector. An anti-FLAG antibody was used to immunoprecipitate the nuclear lysates. Data are presented as the means ± SD. ns , p > 0.05, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: One to two micrograms of primary antibodies were added to the remaining lysate and incubated for 6 h at 4 °C with slow shaking; these antibodies included anti-lamin A/C rabbit polyclonal antibody (10298-1-AP, Proteintech, Wuhan, China), anti-phospho-lamin A/C (S22) antibody (2026, CST), anti-phospho-lamin A/C (S392) antibody (ab58528, Abcam), anti-AMPK alpha 1 antibody (5831, CST, Danvers, MA, USA), and anti-phospho-AMPK alpha (T172) antibody (2535S, CST).

Techniques: Expressing, Negative Control, Transfection, Plasmid Preparation

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: Abnormalities in Lamin A/C function trigger AMPKα activation. ( A ) Mutations in the LMNA structure and sequence are shown: D203N is located in the rod domain and R482W, G465D, and T528R are located in the LTD. Western blotting was performed to detect changes in lamin A/C, pLamin A/C, AMPKα, and pAMPKα-T172 levels upon the rescue of lamin A/C expression via the transfection of the LMNC cDNA, LMNA-GFP cDNA and LMNA mutants, and the corresponding semiquantitative data obtained after GAPDH normalization are shown. ( B ) TG and lactate synthesis were quantified in the different transfected groups ( n = 3). ( C ) Western blotting was performed to assess concentration-dependent changes in lamin A/C, pLamin A/C, AMPKα, pAMPKα, pACC1 and ACC1 levels in 7701, HepG2 and MHCC97-H cells treated with lonafarnib. The corresponding quantitative data were obtained by standardization with GAPDH. Data are presented as the means of at least three independent experiments ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: One to two micrograms of primary antibodies were added to the remaining lysate and incubated for 6 h at 4 °C with slow shaking; these antibodies included anti-lamin A/C rabbit polyclonal antibody (10298-1-AP, Proteintech, Wuhan, China), anti-phospho-lamin A/C (S22) antibody (2026, CST), anti-phospho-lamin A/C (S392) antibody (ab58528, Abcam), anti-AMPK alpha 1 antibody (5831, CST, Danvers, MA, USA), and anti-phospho-AMPK alpha (T172) antibody (2535S, CST).

Techniques: Activation Assay, Sequencing, Western Blot, Expressing, Transfection, Concentration Assay

Journal: Cells

Article Title: Loss of Mature Lamin A/C Triggers a Shift in Intracellular Metabolic Homeostasis via AMPKα Activation

doi: 10.3390/cells11243988

Figure Lengend Snippet: pLamin A/C is an indicator of increased lipid synthesis. ( A ) Treatment with 25 mM glucose for 12 h promoted lamin A/C phosphorylation and FASN expression in 7701, HepG2 and MHCC97-H cells. Semiquantitative analyses of blots are displayed. ( B ) TG synthesis was quantified ( n = 3). ( C ) Lamin A/C phosphorylation was increased by 25 mM glucose in a time-dependent manner in 7701 and HepG2 cells. Gray values from blots are displayed. ( D ) The inhibition of AMPKα activation was shown in 7701, HepG2 or MHCC97-H cells treated with 25 mM glucose. Semiquantitative analyses of blots are displayed. ( E ) Person’s correlation analysis between pLamin A and FASN or pAMPKα-T172 is shown. Data are shown as the means of at least three independent experiments ± SD. * p < 0.05 and ** p < 0.01.

Article Snippet: One to two micrograms of primary antibodies were added to the remaining lysate and incubated for 6 h at 4 °C with slow shaking; these antibodies included anti-lamin A/C rabbit polyclonal antibody (10298-1-AP, Proteintech, Wuhan, China), anti-phospho-lamin A/C (S22) antibody (2026, CST), anti-phospho-lamin A/C (S392) antibody (ab58528, Abcam), anti-AMPK alpha 1 antibody (5831, CST, Danvers, MA, USA), and anti-phospho-AMPK alpha (T172) antibody (2535S, CST).

Techniques: Expressing, Inhibition, Activation Assay

Journal: International Journal of Biological Sciences

Article Title: URI1 suppresses irradiation-induced reactive oxygen species (ROS) by activating autophagy in hepatocellular carcinoma cells

doi: 10.7150/ijbs.55689

Figure Lengend Snippet: URI1 induces autophagy by activating AMPK. (A) Activation of AMPK induces autophagy in HepG2 cells. HepG2 cells were treated with AICAR (1mM) for 6 h and HCQ (50μM) for 12 h. MAP1LC3-II was analyzed by western blot. GAPDH was used as a loading control. AICAR was used as an activator of AMPK. (B) Activation of AMPK induces phosphorylation of FOXO3 at the Ser253 residue. HepG2 cells were treated with AICAR (1mM) or DOX (10μM) for 6 h. FOXO3 was analyzed by western blot for p-FOXO3 (Ser253) and p-FOXO3 (Ser318/321). GAPDH was used as a loading control. AICAR was used as an activator of AMPK. DOX was used as an inhibitor of AMPK. (C) URI1 induces phosphorylation of AMPK at the Thr172 residue and phosphorylation of FOXO3 at the Ser253 residue. HepG2 cells with stable interference (URI1i) or overexpression (URI1o) of URI1 were treated with AICAR (1mM) for 6 h. AMPK was analyzed by western blot for p-AMPKα (Thr172). FOXO3 was analyzed by western blot for p-FOXO3 (Ser253) and p-FOXO3 (Ser318/321). GAPDH was used as a loading control. (D) The ratio of p-AMPKα (Thr172) to AMPKα and the ratio of p-FOXO3(Ser253) to FOXO3 were quantified by using Image J 1.80 software. Representative results of 3 independent experiments are shown. (E) Depletion of URI1 decreases the phosphorylation of AMPK and FOXO3 under the condition of irradiation. HepG2 cells were separately treated with URI1i plasmid or AICAR or DOX and then irradiation for 2 h. AMPK was analyzed by western blot for p-AMPKα (Thr172). FOXO3 was analyzed by western blot for p-FOXO3 (Ser253). GAPDH was used as a loading control. (F) Activation of AMPK decreases irradiation-induced ROS. HepG2 cells were treated with AICAR (1mM) for 6h followed by irradiation for 2 h. Right panel: ROS were analyzed by microplate reader. Values are representative results of 3 independent experiments. ****p<0.0001. Left panel: Western blot showed the phosphorylation of AMPK.

Article Snippet: The following antibodies were used: rabbit anti-MAP1LC3B (3868), rabbit anti-phospho AMPK alpha (Thr172) (2535) and rabbit anti-phospho FOXO3 Ser318/321 (9465) were obtained from CST (Cell Signaling Technology).

Techniques: Activation Assay, Western Blot, Over Expression, Software, Irradiation, Plasmid Preparation