Structured Review

Cell Signaling Technology Inc ampkα
AMPK activity and mitochondrial density in skeletal muscle in young and old WT and MCAT mice. (A) Representative immunoblots for each group and the ratio of p-AMPK T172 to <t>AMPKα,</t> the ratio of p-ACC2 Ser212 and PGC1α to actin in quadriceps
Ampkα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ampkα/product/Cell Signaling Technology Inc
Average 99 stars, based on 55 article reviews
Price from $9.99 to $1999.99
ampkα - by Bioz Stars, 2020-04
99/100 stars

Images

1) Product Images from "Targeted Expression of Catalase to Mitochondria Prevents Age-Associated Reductions in Mitochondrial Function and Insulin Resistance"

Article Title: Targeted Expression of Catalase to Mitochondria Prevents Age-Associated Reductions in Mitochondrial Function and Insulin Resistance

Journal: Cell metabolism

doi: 10.1016/j.cmet.2010.11.004

AMPK activity and mitochondrial density in skeletal muscle in young and old WT and MCAT mice. (A) Representative immunoblots for each group and the ratio of p-AMPK T172 to AMPKα, the ratio of p-ACC2 Ser212 and PGC1α to actin in quadriceps
Figure Legend Snippet: AMPK activity and mitochondrial density in skeletal muscle in young and old WT and MCAT mice. (A) Representative immunoblots for each group and the ratio of p-AMPK T172 to AMPKα, the ratio of p-ACC2 Ser212 and PGC1α to actin in quadriceps

Techniques Used: Activity Assay, Mouse Assay, Western Blot

2) Product Images from "AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation"

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

Journal: Oncotarget

doi: 10.18632/oncotarget.7404

Disruption of AMPKα1(S173) increases cell death during glucose starvation in hepatocarcinoma derived cells C3A cells stably expressing either AMPKα1(S173C) (S173C), AMPKα1(S173D) (S173D) or wild type AMPKα1(S173) (WT) were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. After 12 h of glucose deprivation cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172), AMPKα and Puma. α Tubulin was used as loading control. The blots in the picture are representative of 3 independent experiments. B. Cells attached to microplates were cultured for 0, 24, 48 and 72 h. MTT assay was performed as described in Materials and Methods . Results are presented as the percentage of the absorbance of the cells at 0 h. C. After 36 h cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent fold increase respect to WT controls (WT C) in the proportion of cells undergoing apoptosis (Annexin V positive). WT C apoptotic cells (mean value): 9%. Values represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: Disruption of AMPKα1(S173) increases cell death during glucose starvation in hepatocarcinoma derived cells C3A cells stably expressing either AMPKα1(S173C) (S173C), AMPKα1(S173D) (S173D) or wild type AMPKα1(S173) (WT) were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. After 12 h of glucose deprivation cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172), AMPKα and Puma. α Tubulin was used as loading control. The blots in the picture are representative of 3 independent experiments. B. Cells attached to microplates were cultured for 0, 24, 48 and 72 h. MTT assay was performed as described in Materials and Methods . Results are presented as the percentage of the absorbance of the cells at 0 h. C. After 36 h cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent fold increase respect to WT controls (WT C) in the proportion of cells undergoing apoptosis (Annexin V positive). WT C apoptotic cells (mean value): 9%. Values represent the mean ± SEM of 3 experiments. * P

Techniques Used: Derivative Assay, Stable Transfection, Expressing, Incubation, Western Blot, Cell Culture, MTT Assay, Staining, Flow Cytometry, Cytometry

AMPK and PKA activation during glucose restriction C3A cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). Cell lysates were obtained and analyzed by western blotting. A. Protein levels of P-AMPKα(T172) and AMPKα were detected at 2 and 6 h of glucose deprivation. B. Phosphorylated PKA substrates (RXXS/T-P residues) were detected after 2 h of treatments. Cells incubated with 100 μM dbcAMP (dbcAMP) were used as positive control of PKA phosphorylation. Bars represent band densities of PKA substrates weighted 37, 80 and 140 kDa. α Tubulin was used as loading control. Values represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: AMPK and PKA activation during glucose restriction C3A cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). Cell lysates were obtained and analyzed by western blotting. A. Protein levels of P-AMPKα(T172) and AMPKα were detected at 2 and 6 h of glucose deprivation. B. Phosphorylated PKA substrates (RXXS/T-P residues) were detected after 2 h of treatments. Cells incubated with 100 μM dbcAMP (dbcAMP) were used as positive control of PKA phosphorylation. Bars represent band densities of PKA substrates weighted 37, 80 and 140 kDa. α Tubulin was used as loading control. Values represent the mean ± SEM of 3 experiments. * P

Techniques Used: Activation Assay, Incubation, Western Blot, Positive Control

Effects of AMPK knock down on cell cycle progression and cell death during glucose deprivation in liver cancer cells C3A (A, B, C) HuH-7 and SK-Hep-1 (D) cells were transfected with a siRNA specifically targeted to AMPKα1 mRNA (AMPK KD), or with a siRNA control (Control), and allowed to grow for 48 hours. A. AMPKα expression was analyzed by western blot, in Control and AMPK KD cells. α Tubulin was used as loading control. In panels B, C and D control and AMPK KD cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0) during 24 h. B. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The figure shows typical outputs of the cytometric analysis and percentages of C3A cells in the different phases of the cell cycle, an experiment representative of 3 independent experiments. C. C3A cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent increase in the percentages of cells undergoing apoptosis (Annexin V positive) relative to controls (C). Control apoptotic cells (mean value): 8%. Values represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: Effects of AMPK knock down on cell cycle progression and cell death during glucose deprivation in liver cancer cells C3A (A, B, C) HuH-7 and SK-Hep-1 (D) cells were transfected with a siRNA specifically targeted to AMPKα1 mRNA (AMPK KD), or with a siRNA control (Control), and allowed to grow for 48 hours. A. AMPKα expression was analyzed by western blot, in Control and AMPK KD cells. α Tubulin was used as loading control. In panels B, C and D control and AMPK KD cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0) during 24 h. B. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The figure shows typical outputs of the cytometric analysis and percentages of C3A cells in the different phases of the cell cycle, an experiment representative of 3 independent experiments. C. C3A cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent increase in the percentages of cells undergoing apoptosis (Annexin V positive) relative to controls (C). Control apoptotic cells (mean value): 8%. Values represent the mean ± SEM of 3 experiments. * P

Techniques Used: Transfection, Expressing, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry

Regulation of AMPK activation by PKA phosphorylation in hepatic cancer cells C3A cells were incubated for 36 h (A) and 24 h (B) with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. C and Glc0 cells were incubated in the absence or presence of 100 μM dbcAMP (dbcAMP) or/and 1 mM AICAR (AICAR). Cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172) and AMPKα. β Actin was used as loading control. Bars represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: Regulation of AMPK activation by PKA phosphorylation in hepatic cancer cells C3A cells were incubated for 36 h (A) and 24 h (B) with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. C and Glc0 cells were incubated in the absence or presence of 100 μM dbcAMP (dbcAMP) or/and 1 mM AICAR (AICAR). Cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172) and AMPKα. β Actin was used as loading control. Bars represent the mean ± SEM of 3 experiments. * P

Techniques Used: Activation Assay, Incubation, Western Blot

3) Product Images from "Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis"

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis

Journal: Molecules

doi: 10.3390/molecules170911216

Representative Western blot analyses of p-AMPKβ and AMPKβ ( A ), p-AMPKα and AMPKα ( B ) in rat sciatic nerves. Protein expression was analyzed by western blot, and bands were quantified by densitometry. β-Actin was used as a loading control and for relative quantification in all cases. # p
Figure Legend Snippet: Representative Western blot analyses of p-AMPKβ and AMPKβ ( A ), p-AMPKα and AMPKα ( B ) in rat sciatic nerves. Protein expression was analyzed by western blot, and bands were quantified by densitometry. β-Actin was used as a loading control and for relative quantification in all cases. # p

Techniques Used: Western Blot, Expressing

4) Product Images from "Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation"

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00098

Knockdown of AMPK by siRNA attenuated BA-mediated microglia polarization in BV-2 microglial cells. Cells were first transfected with 40 nM AMPKα siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of AMPKα protein. (B,C) AMPKα knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) AMPKα knockdown attenuated BA-enhanced IL-10 release and mRNA expression of CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P
Figure Legend Snippet: Knockdown of AMPK by siRNA attenuated BA-mediated microglia polarization in BV-2 microglial cells. Cells were first transfected with 40 nM AMPKα siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of AMPKα protein. (B,C) AMPKα knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) AMPKα knockdown attenuated BA-enhanced IL-10 release and mRNA expression of CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P

Techniques Used: Transfection, Expressing, Inhibition

5) Product Images from "Cardioprotection by combination of three compounds from ShengMai preparations in mice with myocardial ischemia/reperfusion injury through AMPK activation-mediated mitochondrial fission"

Article Title: Cardioprotection by combination of three compounds from ShengMai preparations in mice with myocardial ischemia/reperfusion injury through AMPK activation-mediated mitochondrial fission

Journal: Scientific Reports

doi: 10.1038/srep37114

GRS inhibited mitochondrial fission with regulation of AMPK. ( A ) H9c2 cardiomyocytes were treated with GRS at the concentration of 0.1–10 μg/mL and then exposed to hypoxia of 6 h followed by 1 h reoxygenation. AMPKα and p-AMPKα expression were detected by western blot. (B) GRS and AICAR incubated with H/R injury in H9c2 cardiomyocytes transfected with AMPKα or control scrambled siRNAs. AMPKα, Drp1 and p-Drp1 expression were detected by western blot. (C) Mitochondrial fission was detected by Mito Tracker Red with confocal microscopy (Bar = 10 μm). # P
Figure Legend Snippet: GRS inhibited mitochondrial fission with regulation of AMPK. ( A ) H9c2 cardiomyocytes were treated with GRS at the concentration of 0.1–10 μg/mL and then exposed to hypoxia of 6 h followed by 1 h reoxygenation. AMPKα and p-AMPKα expression were detected by western blot. (B) GRS and AICAR incubated with H/R injury in H9c2 cardiomyocytes transfected with AMPKα or control scrambled siRNAs. AMPKα, Drp1 and p-Drp1 expression were detected by western blot. (C) Mitochondrial fission was detected by Mito Tracker Red with confocal microscopy (Bar = 10 μm). # P

Techniques Used: Concentration Assay, Expressing, Western Blot, Incubation, Transfection, Confocal Microscopy

6) Product Images from "SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants"

Article Title: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Journal: eLife

doi: 10.7554/eLife.05828

Characterization of the akin10 mutant line GABI_579E09. (A‐D) Mapping of the T‐DNA insertion site. (A) The top scheme represents the genomic locus of AKIN10 (AT3G01090). UTRs are grey, exons are green boxes. The first exon, which is only present in splicing form 2 (sf2), is shown in light green. By sequencing of the flanking regions the T‐DNA insertion was mapped to position 2583‐2593 (11pb deletion), just before the last exon. Primers used for the genotyping PCR shown in (B) are indicated. The scheme below represents the AKIN10 protein with the kinase domain (KD) in yellow and the kinase associated (KA) domain in blue. The position of K48 (ATP binding; mutated to M in the inactive version) and T175 (target for SnAK2 in the T‐loop), as well as the approximate binding sites of the antibodies against AKIN10 (Agrisera; AS 10919) and AMPKα‐pT172 (Cell Signaling; #2531) are indicated. (B) Genotyping PCR with primers binding in the AKIN 10 sequence (fwA and rvA) and the T‐DNA left border (LB). (C) The scheme represents the genomic locus and protein of IMS2 (AT5G23020), a potential second T‐DNA insertion site. Sequencing of the T‐DNA flanking region by GABI KAT ( www.gabi.kat.da ) produced a sequence with partial homology to IMS2 . However, this insertion was not confirmed. The proposed position of the T‐DNA is indicated. (D) Genotyping PCR with primers binding in the IMS2 sequence (fwI and rvI) and the sequencing primer (seq) used by GABI KAT. (E) Western blots against AKIN10 in Col‐0 wt and akin10 plants. Antibodies against AKIN10 (top) and AMPKα‐pT172, which recognizes both AKIN10 and AKIN11 phosphorylated in the T‐loop, (bottom) were used. The position of AKIN10 and phospho‐AKIN10/11 is indicated. Left: 5 week‐old plants grown on soil in a 12h/12h light/dark cycle. Total proteins were extracted from rosettes harvested after 6h of light (L) or extended night (EN). Right: 2 week‐old seedlings in liquid ½ MS medium. Total proteins were extracted from seedlings treated with 6h of extended night in the presence (+) or absence (‐) of 1% sucrose. (F) Comparison of 8 week‐old wt and akin10 plants. The plants were grown under short day conditions (8h/16h light/dark). The number of leaves, fresh weight (FW), dry weight (DW), and the ratio between FW and DW was determined. The bars represent the mean ± SD of 19‐20 replicates. T‐tests did not produce significant differences between wt and mutant. (G) Mild flowering phenotype of akin10 . Col‐0 wt and akin10 plants were grown under SD conditions and the number of leaves at bolting time were counted. The bars represent the mean ± SD of 15 and 11 replicates, respectively. The p‐value of an unpaired t ‐test with unequal variance was
Figure Legend Snippet: Characterization of the akin10 mutant line GABI_579E09. (A‐D) Mapping of the T‐DNA insertion site. (A) The top scheme represents the genomic locus of AKIN10 (AT3G01090). UTRs are grey, exons are green boxes. The first exon, which is only present in splicing form 2 (sf2), is shown in light green. By sequencing of the flanking regions the T‐DNA insertion was mapped to position 2583‐2593 (11pb deletion), just before the last exon. Primers used for the genotyping PCR shown in (B) are indicated. The scheme below represents the AKIN10 protein with the kinase domain (KD) in yellow and the kinase associated (KA) domain in blue. The position of K48 (ATP binding; mutated to M in the inactive version) and T175 (target for SnAK2 in the T‐loop), as well as the approximate binding sites of the antibodies against AKIN10 (Agrisera; AS 10919) and AMPKα‐pT172 (Cell Signaling; #2531) are indicated. (B) Genotyping PCR with primers binding in the AKIN 10 sequence (fwA and rvA) and the T‐DNA left border (LB). (C) The scheme represents the genomic locus and protein of IMS2 (AT5G23020), a potential second T‐DNA insertion site. Sequencing of the T‐DNA flanking region by GABI KAT ( www.gabi.kat.da ) produced a sequence with partial homology to IMS2 . However, this insertion was not confirmed. The proposed position of the T‐DNA is indicated. (D) Genotyping PCR with primers binding in the IMS2 sequence (fwI and rvI) and the sequencing primer (seq) used by GABI KAT. (E) Western blots against AKIN10 in Col‐0 wt and akin10 plants. Antibodies against AKIN10 (top) and AMPKα‐pT172, which recognizes both AKIN10 and AKIN11 phosphorylated in the T‐loop, (bottom) were used. The position of AKIN10 and phospho‐AKIN10/11 is indicated. Left: 5 week‐old plants grown on soil in a 12h/12h light/dark cycle. Total proteins were extracted from rosettes harvested after 6h of light (L) or extended night (EN). Right: 2 week‐old seedlings in liquid ½ MS medium. Total proteins were extracted from seedlings treated with 6h of extended night in the presence (+) or absence (‐) of 1% sucrose. (F) Comparison of 8 week‐old wt and akin10 plants. The plants were grown under short day conditions (8h/16h light/dark). The number of leaves, fresh weight (FW), dry weight (DW), and the ratio between FW and DW was determined. The bars represent the mean ± SD of 19‐20 replicates. T‐tests did not produce significant differences between wt and mutant. (G) Mild flowering phenotype of akin10 . Col‐0 wt and akin10 plants were grown under SD conditions and the number of leaves at bolting time were counted. The bars represent the mean ± SD of 15 and 11 replicates, respectively. The p‐value of an unpaired t ‐test with unequal variance was

Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Binding Assay, Produced, Western Blot, Mass Spectrometry

7) Product Images from "Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles"

Article Title: Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2017.00330

Undercarboxylated osteocalcin (ucOC) effects on the phosphorylation of ERK2 and AMPKα. The phosphorylation levels, total expression levels, and phospho/total ratio levels of ERK2 (A,B) and AMPKα (C,D) of Extensor digitorum longus (EDL) and soleus samples treated with Krebs–Henseleit buffer control and ucOC (3 and 30 ng mL −1 , N = 9–10 for each dose) were examined. * P ≤ 0.05 and ** P ≤ 0.01 paired samples from the same animal ( t -test).
Figure Legend Snippet: Undercarboxylated osteocalcin (ucOC) effects on the phosphorylation of ERK2 and AMPKα. The phosphorylation levels, total expression levels, and phospho/total ratio levels of ERK2 (A,B) and AMPKα (C,D) of Extensor digitorum longus (EDL) and soleus samples treated with Krebs–Henseleit buffer control and ucOC (3 and 30 ng mL −1 , N = 9–10 for each dose) were examined. * P ≤ 0.05 and ** P ≤ 0.01 paired samples from the same animal ( t -test).

Techniques Used: Expressing

8) Product Images from "Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells"

Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells

Journal: Bone Research

doi: 10.1038/s41413-018-0029-4

ATP-mediated AMPKα pathway inactivation contributes to MSC dysfunction. a Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP. Intracellular ATP concentrations and expression levels of AMPKα and p-AMPKα were examined 0, 1, 3, 6 and 12 h after treatment. b Expression levels of AMPKα and p-AMPKα in the Alpl +/+ and Alpl +/- MSCs were examined by western blotting. c Expression levels of AMPKα, p-AMPKα, ACC and p-ACC in Alpl +/+ and Alpl +/- MSCs transfected with lentivirus were analyzed by western blotting. d Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP with or without 50 μmol⋅L –1 EIPA and 100 μmol⋅L –1 suramin, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. e 293T cells were treated with medium of Alpl +/+ and Alpl +/- MSCs, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. f-h Downregulated AMKPα expression in Alpl +/+ MSCs and treatment with or without 10 μmol⋅L –1 ATP. Ageing-specific genes were analyzed at 48 h by western blotting. Alizarin Red and Oil Red O staining and quantifications were performed on day 21 and day 14 after the osteogenic/adipogenic induction (OS/AD). Expression levels of Runx2, OCN and PPAR-γ were examined by western blotting on day 7 after induction. Scale bars, 100 μm. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. ** P
Figure Legend Snippet: ATP-mediated AMPKα pathway inactivation contributes to MSC dysfunction. a Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP. Intracellular ATP concentrations and expression levels of AMPKα and p-AMPKα were examined 0, 1, 3, 6 and 12 h after treatment. b Expression levels of AMPKα and p-AMPKα in the Alpl +/+ and Alpl +/- MSCs were examined by western blotting. c Expression levels of AMPKα, p-AMPKα, ACC and p-ACC in Alpl +/+ and Alpl +/- MSCs transfected with lentivirus were analyzed by western blotting. d Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP with or without 50 μmol⋅L –1 EIPA and 100 μmol⋅L –1 suramin, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. e 293T cells were treated with medium of Alpl +/+ and Alpl +/- MSCs, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. f-h Downregulated AMKPα expression in Alpl +/+ MSCs and treatment with or without 10 μmol⋅L –1 ATP. Ageing-specific genes were analyzed at 48 h by western blotting. Alizarin Red and Oil Red O staining and quantifications were performed on day 21 and day 14 after the osteogenic/adipogenic induction (OS/AD). Expression levels of Runx2, OCN and PPAR-γ were examined by western blotting on day 7 after induction. Scale bars, 100 μm. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. ** P

Techniques Used: Expressing, Western Blot, Transfection, Staining

Metformin treatment prevents bone ageing in Alpl +/- mice by rescuing the impaired function of MSCs. We injected 60 mg⋅kg –1 metformin into the femoral bone marrow cavity of 4-month-old Alpl +/- mice every 2 weeks for 1 month (total of two injections), and NaCl was used as a control. a Expression levels of p-AMPKα and p-ACC in MSCs from three groups were analyzed by western blotting. b Immunostaining of Ki67, γH2AX and LAP2β in MSCs from control and metformin-treated mice. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs were examined by western blotting. d Alizarin Red staining and quantification of mineralized nodules were performed on day 21 after the osteogenic induction (OS) in the MSCs from Alpl +/+ and Alpl +/- mice injected with NaCl or metformin. Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. e Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). Scale bars, 100 μm. PPAR-γ expression was examined on day 7 after induction by western blotting. f Immunostaining analysis showing the expression of p-AMPKα (red) and nuclear staining (blue, DAPI) in the proximal femoral diaphysis. Quantification of p-AMPKα + cells is indicated in the bottom panel. g μCT images and quantification of BMD and BV/TV. Scale bars, 1 mm. h Images of calcein double labeling of trabecular bone with quantification of BFR/BS. Scale bars, 50 μm. i Oil Red O staining images and quantitative analysis of the area of adipose tissue over the total area of the proximal femoral diaphysis. Scale bars, 500 μm. j Expression levels of ageing-specific genes were examined via qRT-PCR. n = 8 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
Figure Legend Snippet: Metformin treatment prevents bone ageing in Alpl +/- mice by rescuing the impaired function of MSCs. We injected 60 mg⋅kg –1 metformin into the femoral bone marrow cavity of 4-month-old Alpl +/- mice every 2 weeks for 1 month (total of two injections), and NaCl was used as a control. a Expression levels of p-AMPKα and p-ACC in MSCs from three groups were analyzed by western blotting. b Immunostaining of Ki67, γH2AX and LAP2β in MSCs from control and metformin-treated mice. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs were examined by western blotting. d Alizarin Red staining and quantification of mineralized nodules were performed on day 21 after the osteogenic induction (OS) in the MSCs from Alpl +/+ and Alpl +/- mice injected with NaCl or metformin. Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. e Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). Scale bars, 100 μm. PPAR-γ expression was examined on day 7 after induction by western blotting. f Immunostaining analysis showing the expression of p-AMPKα (red) and nuclear staining (blue, DAPI) in the proximal femoral diaphysis. Quantification of p-AMPKα + cells is indicated in the bottom panel. g μCT images and quantification of BMD and BV/TV. Scale bars, 1 mm. h Images of calcein double labeling of trabecular bone with quantification of BFR/BS. Scale bars, 50 μm. i Oil Red O staining images and quantitative analysis of the area of adipose tissue over the total area of the proximal femoral diaphysis. Scale bars, 500 μm. j Expression levels of ageing-specific genes were examined via qRT-PCR. n = 8 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

Techniques Used: Mouse Assay, Injection, Expressing, Western Blot, Immunostaining, Staining, Labeling, Quantitative RT-PCR

Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway. a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67 + , γH2AX + and LAP2β + is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP MSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti- ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mM metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
Figure Legend Snippet: Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway. a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67 + , γH2AX + and LAP2β + is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP MSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti- ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mM metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

Techniques Used: Staining, Immunostaining, Expressing, Western Blot, Concentration Assay, Radioactivity, Plasmid Preparation, Transduction, shRNA

9) Product Images from "Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma"

Article Title: Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma

Journal: British Journal of Cancer

doi: 10.1038/s41416-018-0283-7

EL-4 cells treated with 0 µM leucine experience severe reduction in oxygen consumption, ATP production, and mTORC1 pathway. a Oxygen consumption rate (OCR) as measured by the XF24 analyser in EL-4 cells treated with increasing concentrations of leucine (0, 190, 380, and 1140 µM Leu) for 48 h. The basal respiration was measured first, followed by addition of oligomycin (Olg) to inhibit ATP synthesis, and addition of uncoupler (FCCP) of the oxidative phosphorylation. Lastly, antimycin and rotenone (A/R) were added to inhibit ETC and thus the mitochondrial respiration. The ATP production and maximal respiration were calculated as described. 34 b Western Blotting of AMPKα, S6, and 4EBP-1 in EL-4 cells treated with leucine (0–1140 µM Leu) for 48 h, or NALA (0,10, 20 mM) and rapamycin (0, 100 nM) for 24 h. Image J software was used to calculate the relative ratio (RR) between the phosphorylated and total forms of AMPKα, S6, and 4EBP-1. In all graphs, data represent mean ± SEM, n = 3 independent experiments. ♠ P ≤ 0.05 as compared to 0 µM Leu, ♦ P ≤ 0.05 as compared to 190 µM Leu, ♣ P
Figure Legend Snippet: EL-4 cells treated with 0 µM leucine experience severe reduction in oxygen consumption, ATP production, and mTORC1 pathway. a Oxygen consumption rate (OCR) as measured by the XF24 analyser in EL-4 cells treated with increasing concentrations of leucine (0, 190, 380, and 1140 µM Leu) for 48 h. The basal respiration was measured first, followed by addition of oligomycin (Olg) to inhibit ATP synthesis, and addition of uncoupler (FCCP) of the oxidative phosphorylation. Lastly, antimycin and rotenone (A/R) were added to inhibit ETC and thus the mitochondrial respiration. The ATP production and maximal respiration were calculated as described. 34 b Western Blotting of AMPKα, S6, and 4EBP-1 in EL-4 cells treated with leucine (0–1140 µM Leu) for 48 h, or NALA (0,10, 20 mM) and rapamycin (0, 100 nM) for 24 h. Image J software was used to calculate the relative ratio (RR) between the phosphorylated and total forms of AMPKα, S6, and 4EBP-1. In all graphs, data represent mean ± SEM, n = 3 independent experiments. ♠ P ≤ 0.05 as compared to 0 µM Leu, ♦ P ≤ 0.05 as compared to 190 µM Leu, ♣ P

Techniques Used: Western Blot, Software

a Organ hypertrophy in BCATmKO mice. Heart, kidney, spleen, and muscle (gastric and soleus) weights are expressed in grams (g) and are adjusted for body weight (25 g). b Western blotting of S6 and AMPKα from tumour tissues. Representative protein images from three WT (WT1-3) and BCATmKO (KO1-3) mice are shown. Image J software was used to calculate the relative band ratio between the phosphorylated (P) and total forms of S6 and AMPKα. In all graphs, WT and BCATmKO mice were challenged with EL-4 cells as described in Fig. 1 and organs and tissues were harvested in the end of the tumour study (day 13). Data represent mean ± SEM, n = 6–9, females, age 12–15 weeks, ♣ P ≤ 0.05 as compared to tumour-injected WT mice; * ♠ P ≤ 0.05 as compared to vehicle WT mice
Figure Legend Snippet: a Organ hypertrophy in BCATmKO mice. Heart, kidney, spleen, and muscle (gastric and soleus) weights are expressed in grams (g) and are adjusted for body weight (25 g). b Western blotting of S6 and AMPKα from tumour tissues. Representative protein images from three WT (WT1-3) and BCATmKO (KO1-3) mice are shown. Image J software was used to calculate the relative band ratio between the phosphorylated (P) and total forms of S6 and AMPKα. In all graphs, WT and BCATmKO mice were challenged with EL-4 cells as described in Fig. 1 and organs and tissues were harvested in the end of the tumour study (day 13). Data represent mean ± SEM, n = 6–9, females, age 12–15 weeks, ♣ P ≤ 0.05 as compared to tumour-injected WT mice; * ♠ P ≤ 0.05 as compared to vehicle WT mice

Techniques Used: Mouse Assay, Western Blot, Software, Injection

10) Product Images from "Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation"

Article Title: Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2017.00134

Acanthoic acid increases the LXRs activities and activates the Sirt1/LKB1/AMPK/ACC signaling pathways in EtOH/LPS stimulated AML-12 cell. AML-12 cells were pretreated with AA (5, 10, 20 μM), or AICAR (500 μM), or GW3965 (1 μM), or SRT1720 (6 μM), or Nicotinamide (20 mM) for 2 h, respectively, and then following treated with EtOH (200 mM) and LPS (10 ng/ml) for additional 48 h. (A) Representative western blot analysis of Sirt1, p-LKB1, p-ACC, PPARα, and PPARγ was normalized based on the internal control β-actin. (B) Representative western blot analysis of p-AMPKα, p-AMPKβ, total AMPKα and AMPKβ. (C) Representative western blot analysis of LXRα and LXRβ was normalized based on the internal control GAPDH. (D) Representative western blot analysis of Sirt1, LXRα and LXRβ was normalized based on the internal control GAPDH. # p
Figure Legend Snippet: Acanthoic acid increases the LXRs activities and activates the Sirt1/LKB1/AMPK/ACC signaling pathways in EtOH/LPS stimulated AML-12 cell. AML-12 cells were pretreated with AA (5, 10, 20 μM), or AICAR (500 μM), or GW3965 (1 μM), or SRT1720 (6 μM), or Nicotinamide (20 mM) for 2 h, respectively, and then following treated with EtOH (200 mM) and LPS (10 ng/ml) for additional 48 h. (A) Representative western blot analysis of Sirt1, p-LKB1, p-ACC, PPARα, and PPARγ was normalized based on the internal control β-actin. (B) Representative western blot analysis of p-AMPKα, p-AMPKβ, total AMPKα and AMPKβ. (C) Representative western blot analysis of LXRα and LXRβ was normalized based on the internal control GAPDH. (D) Representative western blot analysis of Sirt1, LXRα and LXRβ was normalized based on the internal control GAPDH. # p

Techniques Used: Western Blot

Effects of AA and acute ethanol challenge on the Sirt1 pathway and the phosphorylation of LKB1, AMPK and ACC. (A) Representative western blot analysis of Sirt1 in the cytoplasm and the nucleus was normalized based on the internal control β-actin or GAPDH; (B) Representative western blot analysis of p-LKB1 and total LKB1; (C) Representative western blot analysis of p-AMPKα, p-AMPKβ, and total AMPKα and AMPKβ; (D) Representative western blot analysis of p-ACC and total ACC. # p
Figure Legend Snippet: Effects of AA and acute ethanol challenge on the Sirt1 pathway and the phosphorylation of LKB1, AMPK and ACC. (A) Representative western blot analysis of Sirt1 in the cytoplasm and the nucleus was normalized based on the internal control β-actin or GAPDH; (B) Representative western blot analysis of p-LKB1 and total LKB1; (C) Representative western blot analysis of p-AMPKα, p-AMPKβ, and total AMPKα and AMPKβ; (D) Representative western blot analysis of p-ACC and total ACC. # p

Techniques Used: Western Blot

11) Product Images from "Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation"

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddu286

Impaired phosphorylation of AMPKα at T172 under FLCN deficiency. ( A ) IGF-1 and insulin in mouse serum were measured using ELISA and radioimmunoassay (RIA), respectively. There was no significant difference between Flcn f/f, CKM-Cre ( Flcn KO) mice
Figure Legend Snippet: Impaired phosphorylation of AMPKα at T172 under FLCN deficiency. ( A ) IGF-1 and insulin in mouse serum were measured using ELISA and radioimmunoassay (RIA), respectively. There was no significant difference between Flcn f/f, CKM-Cre ( Flcn KO) mice

Techniques Used: Enzyme-linked Immunosorbent Assay, RIA Assay, Mouse Assay

12) Product Images from "Inflammation in Response to n3 Fatty Acids in a Porcine Obesity Model"

Article Title: Inflammation in Response to n3 Fatty Acids in a Porcine Obesity Model

Journal: Comparative Medicine

doi:

(A) Relative serum adiponectin and (B) AMPKα, (C) Thr 172 AMPKα, and (D) Thr 172 AMPKα:AMPKα of LD Ossabaw swine fed the low-fat control (LFC), high-fat palm oil (HFP), or high-fat palm oil plus n3 fatty acids (HFPn3) diet.
Figure Legend Snippet: (A) Relative serum adiponectin and (B) AMPKα, (C) Thr 172 AMPKα, and (D) Thr 172 AMPKα:AMPKα of LD Ossabaw swine fed the low-fat control (LFC), high-fat palm oil (HFP), or high-fat palm oil plus n3 fatty acids (HFPn3) diet.

Techniques Used:

13) Product Images from "AMP-Activated Protein Kinase Regulates Intraocular Pressure, Extracellular Matrix, and Cytoskeleton in Trabecular Meshwork"

Article Title: AMP-Activated Protein Kinase Regulates Intraocular Pressure, Extracellular Matrix, and Cytoskeleton in Trabecular Meshwork

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.13-12755

Adenoviral transfer of a dominant negative form of the AMPKα subunit (ad.DN.AMPKα) increases matricellular and ECM expression, decreases the phospho-total RhoA ratio (Ser188), and increases F-actin cytoskeletal staining and disarray. ( A ) Representative immunoblots of ECM proteins from CM of human TM cells treated for 66 hours with null adenoviral vector (ad.null) versus ad.DN.AMPKα at 25 MOI. ( B ) Mean ± SEM integrated band intensities calculated from those immunoblots (* P
Figure Legend Snippet: Adenoviral transfer of a dominant negative form of the AMPKα subunit (ad.DN.AMPKα) increases matricellular and ECM expression, decreases the phospho-total RhoA ratio (Ser188), and increases F-actin cytoskeletal staining and disarray. ( A ) Representative immunoblots of ECM proteins from CM of human TM cells treated for 66 hours with null adenoviral vector (ad.null) versus ad.DN.AMPKα at 25 MOI. ( B ) Mean ± SEM integrated band intensities calculated from those immunoblots (* P

Techniques Used: Dominant Negative Mutation, Expressing, Staining, Western Blot, Plasmid Preparation

Treatment with TGF-β2 leads to transient dephosphorylation of AMPKα in human TM cells. Trabecular meshwork cells were lysed at the specified time intervals after treatment with 2.5 ng/mL TGF-β2. ( A ) Representative immunoblots of cell lysates showing detection of p-AMPKα (Thr172) and total AMPKα, with β-actin loading control. Antibodies detect both α1 and α2 isoforms. ( B ) Mean integrated band intensities calculated from above immunoblots. Data expressed as mean phospho-total ratios (normalized to zero time point) ± SEM (* P
Figure Legend Snippet: Treatment with TGF-β2 leads to transient dephosphorylation of AMPKα in human TM cells. Trabecular meshwork cells were lysed at the specified time intervals after treatment with 2.5 ng/mL TGF-β2. ( A ) Representative immunoblots of cell lysates showing detection of p-AMPKα (Thr172) and total AMPKα, with β-actin loading control. Antibodies detect both α1 and α2 isoforms. ( B ) Mean integrated band intensities calculated from above immunoblots. Data expressed as mean phospho-total ratios (normalized to zero time point) ± SEM (* P

Techniques Used: De-Phosphorylation Assay, Western Blot

Treatment with AICAR leads to phosphorylation and activation of AMPKα. Primary cultured human TM cells were lysed at the specified time intervals after treatment with 0.5 mM AICAR. ( A ) Representative immunoblots of cell lysates showing detection of p-AMPKα (Thr172), total AMPKα, p-ACC, and total ACC with β-actin loading control. Antibodies of AMPKα detect both α1 and α2 isoforms. ( B ) Integrated band intensities calculated from above immunoblots. Data expressed as mean phospho-total ratios (normalized to zero time point) ± SEM (* P
Figure Legend Snippet: Treatment with AICAR leads to phosphorylation and activation of AMPKα. Primary cultured human TM cells were lysed at the specified time intervals after treatment with 0.5 mM AICAR. ( A ) Representative immunoblots of cell lysates showing detection of p-AMPKα (Thr172), total AMPKα, p-ACC, and total ACC with β-actin loading control. Antibodies of AMPKα detect both α1 and α2 isoforms. ( B ) Integrated band intensities calculated from above immunoblots. Data expressed as mean phospho-total ratios (normalized to zero time point) ± SEM (* P

Techniques Used: Activation Assay, Cell Culture, Western Blot

In human TM, AMPKα1 and AMPKα2 are expressed. ( A ) Representative immunoblots show detection of p-AMPKα, AMPKα1, and AMPKα2 in cell lysates of primary cultured human TM cells ( n = 4), each at approximately 62 kDa. ( B ) Representative immunofluorescent staining of AMPKα1 and AMPKα2 in sections of adult human cadaveric donor eyes ( n = 4). Nuclei were stained with DAPI. Scale bars : 50 μm.
Figure Legend Snippet: In human TM, AMPKα1 and AMPKα2 are expressed. ( A ) Representative immunoblots show detection of p-AMPKα, AMPKα1, and AMPKα2 in cell lysates of primary cultured human TM cells ( n = 4), each at approximately 62 kDa. ( B ) Representative immunofluorescent staining of AMPKα1 and AMPKα2 in sections of adult human cadaveric donor eyes ( n = 4). Nuclei were stained with DAPI. Scale bars : 50 μm.

Techniques Used: Western Blot, Cell Culture, Staining

14) Product Images from "Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells"

Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells

Journal: Bone Research

doi: 10.1038/s41413-018-0029-4

ATP-mediated AMPKα pathway inactivation contributes to MSC dysfunction. a Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP. Intracellular ATP concentrations and expression levels of AMPKα and p-AMPKα were examined 0, 1, 3, 6 and 12 h after treatment. b Expression levels of AMPKα and p-AMPKα in the Alpl +/+ and Alpl +/- MSCs were examined by western blotting. c Expression levels of AMPKα, p-AMPKα, ACC and p-ACC in Alpl +/+ and Alpl +/- MSCs transfected with lentivirus were analyzed by western blotting. d Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP with or without 50 μmol⋅L –1 EIPA and 100 μmol⋅L –1 suramin, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. e 293T cells were treated with medium of Alpl +/+ and Alpl +/- MSCs, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. f-h Downregulated AMKPα expression in Alpl +/+ MSCs and treatment with or without 10 μmol⋅L –1 ATP. Ageing-specific genes were analyzed at 48 h by western blotting. Alizarin Red and Oil Red O staining and quantifications were performed on day 21 and day 14 after the osteogenic/adipogenic induction (OS/AD). Expression levels of Runx2, OCN and PPAR-γ were examined by western blotting on day 7 after induction. Scale bars, 100 μm. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. ** P
Figure Legend Snippet: ATP-mediated AMPKα pathway inactivation contributes to MSC dysfunction. a Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP. Intracellular ATP concentrations and expression levels of AMPKα and p-AMPKα were examined 0, 1, 3, 6 and 12 h after treatment. b Expression levels of AMPKα and p-AMPKα in the Alpl +/+ and Alpl +/- MSCs were examined by western blotting. c Expression levels of AMPKα, p-AMPKα, ACC and p-ACC in Alpl +/+ and Alpl +/- MSCs transfected with lentivirus were analyzed by western blotting. d Alpl +/+ MSCs were treated with 10 μmol⋅L –1 ATP with or without 50 μmol⋅L –1 EIPA and 100 μmol⋅L –1 suramin, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. e 293T cells were treated with medium of Alpl +/+ and Alpl +/- MSCs, and the expression levels of p-AMPKα and p-ACC were analyzed by western blotting. f-h Downregulated AMKPα expression in Alpl +/+ MSCs and treatment with or without 10 μmol⋅L –1 ATP. Ageing-specific genes were analyzed at 48 h by western blotting. Alizarin Red and Oil Red O staining and quantifications were performed on day 21 and day 14 after the osteogenic/adipogenic induction (OS/AD). Expression levels of Runx2, OCN and PPAR-γ were examined by western blotting on day 7 after induction. Scale bars, 100 μm. n = 6 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. ** P

Techniques Used: Expressing, Western Blot, Transfection, Staining

Metformin treatment prevents bone ageing in Alpl +/- mice by rescuing the impaired function of MSCs. We injected 60 mg⋅kg –1 metformin into the femoral bone marrow cavity of 4-month-old Alpl +/- mice every 2 weeks for 1 month (total of two injections), and NaCl was used as a control. a Expression levels of p-AMPKα and p-ACC in MSCs from three groups were analyzed by western blotting. b Immunostaining of Ki67, γH2AX and LAP2β in MSCs from control and metformin-treated mice. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs were examined by western blotting. d Alizarin Red staining and quantification of mineralized nodules were performed on day 21 after the osteogenic induction (OS) in the MSCs from Alpl +/+ and Alpl +/- mice injected with NaCl or metformin. Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. e Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). Scale bars, 100 μm. PPAR-γ expression was examined on day 7 after induction by western blotting. f Immunostaining analysis showing the expression of p-AMPKα (red) and nuclear staining (blue, DAPI) in the proximal femoral diaphysis. Quantification of p-AMPKα + cells is indicated in the bottom panel. g μCT images and quantification of BMD and BV/TV. Scale bars, 1 mm. h Images of calcein double labeling of trabecular bone with quantification of BFR/BS. Scale bars, 50 μm. i Oil Red O staining images and quantitative analysis of the area of adipose tissue over the total area of the proximal femoral diaphysis. Scale bars, 500 μm. j Expression levels of ageing-specific genes were examined via qRT-PCR. n = 8 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
Figure Legend Snippet: Metformin treatment prevents bone ageing in Alpl +/- mice by rescuing the impaired function of MSCs. We injected 60 mg⋅kg –1 metformin into the femoral bone marrow cavity of 4-month-old Alpl +/- mice every 2 weeks for 1 month (total of two injections), and NaCl was used as a control. a Expression levels of p-AMPKα and p-ACC in MSCs from three groups were analyzed by western blotting. b Immunostaining of Ki67, γH2AX and LAP2β in MSCs from control and metformin-treated mice. Quantification of Ki67 + , γH2AX + and LAP2β + is shown in the right panel. Scale bars: 50 μm. c Expression levels of p16 and p53 in MSCs were examined by western blotting. d Alizarin Red staining and quantification of mineralized nodules were performed on day 21 after the osteogenic induction (OS) in the MSCs from Alpl +/+ and Alpl +/- mice injected with NaCl or metformin. Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. e Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). Scale bars, 100 μm. PPAR-γ expression was examined on day 7 after induction by western blotting. f Immunostaining analysis showing the expression of p-AMPKα (red) and nuclear staining (blue, DAPI) in the proximal femoral diaphysis. Quantification of p-AMPKα + cells is indicated in the bottom panel. g μCT images and quantification of BMD and BV/TV. Scale bars, 1 mm. h Images of calcein double labeling of trabecular bone with quantification of BFR/BS. Scale bars, 50 μm. i Oil Red O staining images and quantitative analysis of the area of adipose tissue over the total area of the proximal femoral diaphysis. Scale bars, 500 μm. j Expression levels of ageing-specific genes were examined via qRT-PCR. n = 8 per group. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

Techniques Used: Mouse Assay, Injection, Expressing, Western Blot, Immunostaining, Staining, Labeling, Quantitative RT-PCR

Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway. a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67 + , γH2AX + and LAP2β + is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP MSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti- ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mM metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P
Figure Legend Snippet: Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway. a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67 + , γH2AX + and LAP2β + is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP MSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ- 32 P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti- ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mM metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. * P

Techniques Used: Staining, Immunostaining, Expressing, Western Blot, Concentration Assay, Radioactivity, Plasmid Preparation, Transduction, shRNA

15) Product Images from "A novel, de novo mutation in the PRKAG2 gene: infantile-onset phenotype and the signaling pathway involved"

Article Title: A novel, de novo mutation in the PRKAG2 gene: infantile-onset phenotype and the signaling pathway involved

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00813.2016

Overexpression of the K475E mutant in H9c2 cells results in AMPK inhibition and hypertrophy. H9c2 cells were stably transfected with human PRKAG2 WT or the K475E mutant construct. A : cells were treated with vehicle or phenformin (1.25 mM) for 1, 4, or 24 h. Representative Western blots with antibody against phospho-AMPKα (T172) or AMPKα are shown. B : densitometric scanning results of the Western blots shown in A . Each bar represents measurement of phospho-AMPKα normalized with AMPKα from 3−4 separate experiments. * P
Figure Legend Snippet: Overexpression of the K475E mutant in H9c2 cells results in AMPK inhibition and hypertrophy. H9c2 cells were stably transfected with human PRKAG2 WT or the K475E mutant construct. A : cells were treated with vehicle or phenformin (1.25 mM) for 1, 4, or 24 h. Representative Western blots with antibody against phospho-AMPKα (T172) or AMPKα are shown. B : densitometric scanning results of the Western blots shown in A . Each bar represents measurement of phospho-AMPKα normalized with AMPKα from 3−4 separate experiments. * P

Techniques Used: Over Expression, Mutagenesis, Inhibition, Stable Transfection, Transfection, Construct, Western Blot

16) Product Images from "G0S2 modulates homeostatic proliferation of naïve CD8+ T cells and inhibits oxidative phosphorylation in mitochondria"

Article Title: G0S2 modulates homeostatic proliferation of naïve CD8+ T cells and inhibits oxidative phosphorylation in mitochondria

Journal: Immunology and cell biology

doi: 10.1038/icb.2015.9

Loss of G0S2 deregulates activation of AMPK and mTOR pathways in CD8 + T cells CD8 + T cells from wild type and G0s2 −/− mice were activated by plate-bound anti-CD23 and anti-CD28 for 12, 24, and 30 hours. Immunoblot analysis of AMPKα (AMP-activated protein kinase α), ACC (Acetyl-CoA carboxylase), S6K (ribosomal protein S6 kinase), Cyclin E, cdk2, phospho-Rb (Ser 780), and tubulin is shown. Data represent three independent experiments.
Figure Legend Snippet: Loss of G0S2 deregulates activation of AMPK and mTOR pathways in CD8 + T cells CD8 + T cells from wild type and G0s2 −/− mice were activated by plate-bound anti-CD23 and anti-CD28 for 12, 24, and 30 hours. Immunoblot analysis of AMPKα (AMP-activated protein kinase α), ACC (Acetyl-CoA carboxylase), S6K (ribosomal protein S6 kinase), Cyclin E, cdk2, phospho-Rb (Ser 780), and tubulin is shown. Data represent three independent experiments.

Techniques Used: Activation Assay, Mouse Assay

17) Product Images from "Uncoupling of the LKB1-AMPK? Energy Sensor Pathway by Growth Factors and Oncogenic BRAFV600E"

Article Title: Uncoupling of the LKB1-AMPK? Energy Sensor Pathway by Growth Factors and Oncogenic BRAFV600E

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004771

Restoration of the LKB1-AMPKα pathway in BRAF V600E melanoma cells induces apoptosis under energy stress conditions. (A) UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (H.G.), or low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for 12 h. Then, Annexin V and PI (propidium iodide) positive cells were analyzed by flow cytometry. Histograms show the result from FACS analysis. Graphs on the right show the percentage of viable and dead cells in a parallel experiment under the same conditions determined by nuclear staining exclusion (Guava-ViaCount). (B) Time course at 4 and 12 hours showing the LKB1-AMPKα pathway status under the same conditions. UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (high glucose H.G.), low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for the times indicated. Fifty micrograms of total protein lysates were separated by SDS-PAGE and same membranes were blotted against the indicated antibodies. All experiments were done at least three times. Representative experiments are shown. (C) UACC903 cells were transfected either with a scramble siRNA or with equimolar amounts of AMPKα1 and AMPKα2 siRNAs for a total concentration of 100 nM. 72 hours after transfection cells were starved in low glucose medium for 6 hours in the presence or absence of 10 µM of U0126. Dead cells were quantified by nuclear staining exclusion (Guava-ViaCount). Western-blots show the levels of p-AMPKα T172 , AMPKα and p-Erk1/2 Thr202/Tyr204 under the different conditions. (D) UACC903 and A375 melanoma cells were grown in complete medium (H.G. cm), serum free high glucose medium (H.G. sf), serum free low glucose medium (L.G.), serum free complete medium plus U0126 10 µM (H.G.+U0126) and low glucose serum free medium plus U0126 10 µM (L.G.+U0126) for 12 hours. The levels of Bim, phospho-Bad and Mcl-1 are showed under the different experimental conditions.
Figure Legend Snippet: Restoration of the LKB1-AMPKα pathway in BRAF V600E melanoma cells induces apoptosis under energy stress conditions. (A) UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (H.G.), or low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for 12 h. Then, Annexin V and PI (propidium iodide) positive cells were analyzed by flow cytometry. Histograms show the result from FACS analysis. Graphs on the right show the percentage of viable and dead cells in a parallel experiment under the same conditions determined by nuclear staining exclusion (Guava-ViaCount). (B) Time course at 4 and 12 hours showing the LKB1-AMPKα pathway status under the same conditions. UACC903, A375 and SKMel28 human melanoma cells were grown in complete medium (high glucose H.G.), low glucose medium (L.G.) with or without 10 µM of the Mek1/2 specific inhibitor U0126 for the times indicated. Fifty micrograms of total protein lysates were separated by SDS-PAGE and same membranes were blotted against the indicated antibodies. All experiments were done at least three times. Representative experiments are shown. (C) UACC903 cells were transfected either with a scramble siRNA or with equimolar amounts of AMPKα1 and AMPKα2 siRNAs for a total concentration of 100 nM. 72 hours after transfection cells were starved in low glucose medium for 6 hours in the presence or absence of 10 µM of U0126. Dead cells were quantified by nuclear staining exclusion (Guava-ViaCount). Western-blots show the levels of p-AMPKα T172 , AMPKα and p-Erk1/2 Thr202/Tyr204 under the different conditions. (D) UACC903 and A375 melanoma cells were grown in complete medium (H.G. cm), serum free high glucose medium (H.G. sf), serum free low glucose medium (L.G.), serum free complete medium plus U0126 10 µM (H.G.+U0126) and low glucose serum free medium plus U0126 10 µM (L.G.+U0126) for 12 hours. The levels of Bim, phospho-Bad and Mcl-1 are showed under the different experimental conditions.

Techniques Used: Flow Cytometry, Cytometry, FACS, Staining, SDS Page, Transfection, Concentration Assay, Western Blot

Inhibition of oncogenic BRAF V600E signaling restores the limited response to metabolic stress of BRAF mutant melanoma cell lines. (A) BRAF mutant melanoma cells have a limited response to energy withdrawal that is restored by U0126 treatment. Fifty micrograms of total lysates from UACC903, A375, SKMel28 and 37-31E melanoma cells grown in serum free high glucose medium (H.G.), serum free low glucose medium (L.G.) or serum free low glucose medium (L.G.) plus 10 µM of U0126 for 4 hours were separated by SDS-PAGE. Western-Blot shows the activation status of proteins in the RAS and LKB1-AMPK-mTOR pathways. (B) U0126 inhibitor treatment does not activate AMPK. UACC903 A375 and SKMel28 melanoma cells were grown in high glucose medium with serum in the absence or presence of 1 µM, 5 µM or 10 µM of U0126. Total protein lysates were subjected to SDS-PAGE. Western-blot shows the phosphorylation state of AMPKα in the presence of different concentrations of U0126. (C) Inhibition of BRAF signaling increases cell response to AICAR. UACC903 and SKMel28 cells were grown in complete medium; cells were treated with AICAR (1 mM) for 4 h in the presence or absence of U0126 (10 µM) inhibitor. p-LKB1 Ser428 , p-AMPK Thr172 , p-Erk1/2 Thr202/Tyr204 , p-ACC Ser79 levels were checked by western blot. (D) Sorafenib treatment and siRNA BRAF knockdown restores the metabolic stress pathway in BRAF mutant melanoma cells. In the left panel, UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of U0126 (10 µM) Western blots show the levels of p-AMPKα T172 , p-LKB1 Ser431 p-Erk1/2 Thr202/Tyr204 and pCREB Ser133 proteins under the different conditions. In the right panel SKMel28 cells were transfected with either a scramble siRNA or BRAF siRNA. 72 hours after transfection, cells were starved either in high glucose (H.G.) or low glucose (L.G.) medium for six hours. Western-blots show the levels of p-AMPKα T172 , p-Erk1/2 Thr202/Tyr204 and BRAF proteins. (E) p90 Rsk inhibitor BI-D1870 (10 µM), does not restore the metabolic stress pathway. UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of BI-D1870 (10 µM). Western blots show the levels of the indicated proteins under the different conditions.
Figure Legend Snippet: Inhibition of oncogenic BRAF V600E signaling restores the limited response to metabolic stress of BRAF mutant melanoma cell lines. (A) BRAF mutant melanoma cells have a limited response to energy withdrawal that is restored by U0126 treatment. Fifty micrograms of total lysates from UACC903, A375, SKMel28 and 37-31E melanoma cells grown in serum free high glucose medium (H.G.), serum free low glucose medium (L.G.) or serum free low glucose medium (L.G.) plus 10 µM of U0126 for 4 hours were separated by SDS-PAGE. Western-Blot shows the activation status of proteins in the RAS and LKB1-AMPK-mTOR pathways. (B) U0126 inhibitor treatment does not activate AMPK. UACC903 A375 and SKMel28 melanoma cells were grown in high glucose medium with serum in the absence or presence of 1 µM, 5 µM or 10 µM of U0126. Total protein lysates were subjected to SDS-PAGE. Western-blot shows the phosphorylation state of AMPKα in the presence of different concentrations of U0126. (C) Inhibition of BRAF signaling increases cell response to AICAR. UACC903 and SKMel28 cells were grown in complete medium; cells were treated with AICAR (1 mM) for 4 h in the presence or absence of U0126 (10 µM) inhibitor. p-LKB1 Ser428 , p-AMPK Thr172 , p-Erk1/2 Thr202/Tyr204 , p-ACC Ser79 levels were checked by western blot. (D) Sorafenib treatment and siRNA BRAF knockdown restores the metabolic stress pathway in BRAF mutant melanoma cells. In the left panel, UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of U0126 (10 µM) Western blots show the levels of p-AMPKα T172 , p-LKB1 Ser431 p-Erk1/2 Thr202/Tyr204 and pCREB Ser133 proteins under the different conditions. In the right panel SKMel28 cells were transfected with either a scramble siRNA or BRAF siRNA. 72 hours after transfection, cells were starved either in high glucose (H.G.) or low glucose (L.G.) medium for six hours. Western-blots show the levels of p-AMPKα T172 , p-Erk1/2 Thr202/Tyr204 and BRAF proteins. (E) p90 Rsk inhibitor BI-D1870 (10 µM), does not restore the metabolic stress pathway. UACC903, A375 and SKMel28 melanoma cells were grown in low glucose serum free medium+/−EGF (50 ng/ml) for 4 h in the presence or absence of BI-D1870 (10 µM). Western blots show the levels of the indicated proteins under the different conditions.

Techniques Used: Inhibition, Mutagenesis, SDS Page, Western Blot, Activation Assay, Transfection

Growth factor treatment and BRAF V600E promotes LKB1-AMPKα disassembly. (A) 293T cells were transiently transfected for 48 h with Flag-LKB1, Flag-LKB1KD (kinase dead) and GST-AMPKα as indicated. Then, cells were treated with 100 ng/ml of EGF for 10 min. Immunocomplexes pulled down with an anti-Flag-resin were separated by SDS-PAGE and proteins present in the complexes were analyzed by western blot. Total lysates show the transfection controls and the response to growth factor treatment. (B) 293T cells were transiently transfected with the constructs indicated. Then, cells were serum starved for 2 h and treated with 100 ng/ml of EGF for 10 min and protein complexes were immunoprecipitated with anti-Flag-resin. Protein complexes were separated by SDS-PAGE. Levels of GST-AMPKα, Flag-LKB1 constructs and the phosphorylation state of LKB1 Ser431 in the complexes are shown. Quantification of the amount of GST-AMPKα normalized to the Flag-LKB1 immunoprecipitated is represented in the graph. Total lysates are shown for control transfection of the different samples. (C) Endogenous LKB1 from UACC903 and A375 melanoma cells growing in low glucose medium (L.G.) with or without 10 µM U0126 was immunoprecipitated. Western-blot from the immunoprecipitated samples was probed against LKB1, AMPKα and p-AMPKα T172 antibodies. On the right, total lysates from SKMel28 melanoma cells growing in complete medium (High Glucose, H.G.) low glucose medium (L.G.) in the presence or absence of 10 µM U0126 were subjected to immunoprecipitation with the anti-p-AMPKα T172 . Samples were separated by SDS-PAGE. Total lysates (T.L.) from low glucose plus U0126 treated cells are showed as a control. Western-Blot of the immunoprecipitated samples was performed against total AMPKα antibody. (D) Hela cells were transfected with Flag-LKB1, GST-AMPKα and myc-BRAF V600E or and empty vector as indicated. Flag-LKB1 was immunoprecipitated and western-blots from immunoprecipitated samples were probed against the indicated antibodies. Graph shows the quantification of the AMPK bound to LKB1.
Figure Legend Snippet: Growth factor treatment and BRAF V600E promotes LKB1-AMPKα disassembly. (A) 293T cells were transiently transfected for 48 h with Flag-LKB1, Flag-LKB1KD (kinase dead) and GST-AMPKα as indicated. Then, cells were treated with 100 ng/ml of EGF for 10 min. Immunocomplexes pulled down with an anti-Flag-resin were separated by SDS-PAGE and proteins present in the complexes were analyzed by western blot. Total lysates show the transfection controls and the response to growth factor treatment. (B) 293T cells were transiently transfected with the constructs indicated. Then, cells were serum starved for 2 h and treated with 100 ng/ml of EGF for 10 min and protein complexes were immunoprecipitated with anti-Flag-resin. Protein complexes were separated by SDS-PAGE. Levels of GST-AMPKα, Flag-LKB1 constructs and the phosphorylation state of LKB1 Ser431 in the complexes are shown. Quantification of the amount of GST-AMPKα normalized to the Flag-LKB1 immunoprecipitated is represented in the graph. Total lysates are shown for control transfection of the different samples. (C) Endogenous LKB1 from UACC903 and A375 melanoma cells growing in low glucose medium (L.G.) with or without 10 µM U0126 was immunoprecipitated. Western-blot from the immunoprecipitated samples was probed against LKB1, AMPKα and p-AMPKα T172 antibodies. On the right, total lysates from SKMel28 melanoma cells growing in complete medium (High Glucose, H.G.) low glucose medium (L.G.) in the presence or absence of 10 µM U0126 were subjected to immunoprecipitation with the anti-p-AMPKα T172 . Samples were separated by SDS-PAGE. Total lysates (T.L.) from low glucose plus U0126 treated cells are showed as a control. Western-Blot of the immunoprecipitated samples was performed against total AMPKα antibody. (D) Hela cells were transfected with Flag-LKB1, GST-AMPKα and myc-BRAF V600E or and empty vector as indicated. Flag-LKB1 was immunoprecipitated and western-blots from immunoprecipitated samples were probed against the indicated antibodies. Graph shows the quantification of the AMPK bound to LKB1.

Techniques Used: Transfection, SDS Page, Western Blot, Construct, Immunoprecipitation, Plasmid Preparation

18) Product Images from "Role of AMP-activated Protein Kinase in Ferritin H Gene Expression by Resveratrol in Human T Cells"

Article Title: Role of AMP-activated Protein Kinase in Ferritin H Gene Expression by Resveratrol in Human T Cells

Journal: Biochemistry

doi: 10.1021/bi400399f

PBMCs were transfected with siRNA or treated with AMPKα inhibitor; control: non-target siRNA, AMPK inhibitor: non-target siRNA + 10 µM Compound C, siAMPKα: AMPKα-targeted siRNA. PBMCs were stimulated with 50 µM
Figure Legend Snippet: PBMCs were transfected with siRNA or treated with AMPKα inhibitor; control: non-target siRNA, AMPK inhibitor: non-target siRNA + 10 µM Compound C, siAMPKα: AMPKα-targeted siRNA. PBMCs were stimulated with 50 µM

Techniques Used: Transfection

Resveratrol-mediated induction of ferritin H and HO-1 mRNA is associated with AMPKα. (A) Jurkat cells were treated with different amounts of resveratrol for 2 h. Nuclear and cytosolic fractions were subjected to Western blotting with antibodies
Figure Legend Snippet: Resveratrol-mediated induction of ferritin H and HO-1 mRNA is associated with AMPKα. (A) Jurkat cells were treated with different amounts of resveratrol for 2 h. Nuclear and cytosolic fractions were subjected to Western blotting with antibodies

Techniques Used: Western Blot

AMPKα-dependent GSK3β phosphorylation at Ser9 is associated with Nrf2-ARE activation. (A, B) Jurkat cells were treated with 30 µM resveratrol for 4 h or 24 h (A). Transfected Jurkat cells with siRNA against AMPKα were treated
Figure Legend Snippet: AMPKα-dependent GSK3β phosphorylation at Ser9 is associated with Nrf2-ARE activation. (A, B) Jurkat cells were treated with 30 µM resveratrol for 4 h or 24 h (A). Transfected Jurkat cells with siRNA against AMPKα were treated

Techniques Used: Activation Assay, Transfection

19) Product Images from "Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells"

Article Title: Glutaminase inhibitor CB-839 synergizes with carfilzomib in resistant multiple myeloma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.16262

PI resistant cells show increased rates of mitochondrial respiration for energy production ( A ) MM.1S and MM.1S BzR cell lines were plated in XF running buffer and exposed to the Seahorse XF Cell Mito Stress Test, consisting of automated treatment with oligomycin, FCCP, and the combination of antimycin A and rotenone at the indicated times. The oxygen consumption rate (OCR) was measured over time using the XF Extracellular Flux Analyzer. A representative full time course of the XF Cell Mito Stress Test is shown. ( B ) Basal OCR (left) and maximum OCR (right) are shown for 4 independent experiments using cells of various passage number that were analyzed as described in (A). For the determination of maximum OCR, three different concentrations of oligomycin (0.05, 0.1, or 1 μM) were used. Note that regardless of the oligomycin concentration used, the MM.1S BzR cells showed significantly higher max OCR. P -values and statistical significance were determined using student's t-test . ( C ) Western blots are shown for untreated MM.1S, MM.1S BzR, U266, and U266 BzR cells [P-AMPKα: phospho-AMP kinase alpha (Thr172); ACC: acetyl-CoA carboxylase (Ser79)]. ( D ) Inherent cellular fluorescence of NAD(P)H was measured in untreated cells. Kinetic data from representative experiments are shown (left). The average NAD(P)H ± S.E. levels over a 100 second timeframe are shown (right). A student's t-test was used to evaluate statistical significance.
Figure Legend Snippet: PI resistant cells show increased rates of mitochondrial respiration for energy production ( A ) MM.1S and MM.1S BzR cell lines were plated in XF running buffer and exposed to the Seahorse XF Cell Mito Stress Test, consisting of automated treatment with oligomycin, FCCP, and the combination of antimycin A and rotenone at the indicated times. The oxygen consumption rate (OCR) was measured over time using the XF Extracellular Flux Analyzer. A representative full time course of the XF Cell Mito Stress Test is shown. ( B ) Basal OCR (left) and maximum OCR (right) are shown for 4 independent experiments using cells of various passage number that were analyzed as described in (A). For the determination of maximum OCR, three different concentrations of oligomycin (0.05, 0.1, or 1 μM) were used. Note that regardless of the oligomycin concentration used, the MM.1S BzR cells showed significantly higher max OCR. P -values and statistical significance were determined using student's t-test . ( C ) Western blots are shown for untreated MM.1S, MM.1S BzR, U266, and U266 BzR cells [P-AMPKα: phospho-AMP kinase alpha (Thr172); ACC: acetyl-CoA carboxylase (Ser79)]. ( D ) Inherent cellular fluorescence of NAD(P)H was measured in untreated cells. Kinetic data from representative experiments are shown (left). The average NAD(P)H ± S.E. levels over a 100 second timeframe are shown (right). A student's t-test was used to evaluate statistical significance.

Techniques Used: Concentration Assay, Western Blot, Fluorescence

20) Product Images from "The hypotensive effect of acute and chronic AMP-activated protein kinase activation in normal and hyperlipidemic mice"

Article Title: The hypotensive effect of acute and chronic AMP-activated protein kinase activation in normal and hyperlipidemic mice

Journal: Vascular Pharmacology

doi: 10.1016/j.vph.2015.07.010

AMPKα expression in aortae of C57BL/6 and ApoE −/− mice treated with AICAR. (A) Blots shown are representative. (B) Phosphorylated and (C) total AMPKα were divided by GAPDH to adjust for protein loading to measure the amount of each form of the enzyme. (D) A ratio of phosphorylated to total AMPKα was calculated to measure the phosphorylation status of the enzyme. *p
Figure Legend Snippet: AMPKα expression in aortae of C57BL/6 and ApoE −/− mice treated with AICAR. (A) Blots shown are representative. (B) Phosphorylated and (C) total AMPKα were divided by GAPDH to adjust for protein loading to measure the amount of each form of the enzyme. (D) A ratio of phosphorylated to total AMPKα was calculated to measure the phosphorylation status of the enzyme. *p

Techniques Used: Expressing, Mouse Assay

21) Product Images from "The Effects of Testosterone Deprivation and Supplementation on Proteasomal and Autophagy Activity in the Skeletal Muscle of the Male Mouse: Differential Effects on High-Androgen Responder and Low-Androgen Responder Muscle Groups"

Article Title: The Effects of Testosterone Deprivation and Supplementation on Proteasomal and Autophagy Activity in the Skeletal Muscle of the Male Mouse: Differential Effects on High-Androgen Responder and Low-Androgen Responder Muscle Groups

Journal: Endocrinology

doi: 10.1210/en.2013-1004

Castration activates AMPKα and PPARδ in the levator ani muscle. Castration was associated with increased AMPKα phosphorylation on threonine 172 (A and B), increased Lkb1 gene expression (C), and increased PPARδ protein
Figure Legend Snippet: Castration activates AMPKα and PPARδ in the levator ani muscle. Castration was associated with increased AMPKα phosphorylation on threonine 172 (A and B), increased Lkb1 gene expression (C), and increased PPARδ protein

Techniques Used: Expressing

Castration inhibits mTOR and activates AMPK in the triceps muscle. (A and B) Western blotting for mTOR. Castration increased TSC2 activation and level (A, C, and D) but did not change significantly the AMPKα-dependent phosphorylation of TSC2 (F).
Figure Legend Snippet: Castration inhibits mTOR and activates AMPK in the triceps muscle. (A and B) Western blotting for mTOR. Castration increased TSC2 activation and level (A, C, and D) but did not change significantly the AMPKα-dependent phosphorylation of TSC2 (F).

Techniques Used: Western Blot, Activation Assay

22) Product Images from "Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase *"

Article Title: Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.143685

Mice with pharmacologic inhibition of catalase activity or acatalasemic mice have increased activation and oxidation of AMPKα subunit in the lungs. A and B , mice were given 50 μl of saline or LPS (1 mg/kg) in 50 μl of saline intratracheally and were treated with ATZ i.p. at 500 mg/kg. ATZ was administered 4 h before intratracheal saline or LPS administration. Lungs were harvested 24 h after treatment of the mice with LPS, and the levels of the AMPKα or AMPKβ subunits, phospho-Thr 172 -AMPKα, ACC, and phospho-Ser 79 -ACC ( p-ACC ), were determined using Western blot analysis. Representative Western blots are shown in A , whereas quantitative analysis of AMPK phosphorylation in lung homogenates is shown in B (mean ± S.D. values were obtained from 3 mice/group; *, p
Figure Legend Snippet: Mice with pharmacologic inhibition of catalase activity or acatalasemic mice have increased activation and oxidation of AMPKα subunit in the lungs. A and B , mice were given 50 μl of saline or LPS (1 mg/kg) in 50 μl of saline intratracheally and were treated with ATZ i.p. at 500 mg/kg. ATZ was administered 4 h before intratracheal saline or LPS administration. Lungs were harvested 24 h after treatment of the mice with LPS, and the levels of the AMPKα or AMPKβ subunits, phospho-Thr 172 -AMPKα, ACC, and phospho-Ser 79 -ACC ( p-ACC ), were determined using Western blot analysis. Representative Western blots are shown in A , whereas quantitative analysis of AMPK phosphorylation in lung homogenates is shown in B (mean ± S.D. values were obtained from 3 mice/group; *, p

Techniques Used: Mouse Assay, Inhibition, Activity Assay, Activation Assay, Western Blot

Putative mechanism of AMPK activation by H 2 O 2 . The AMPKαβγ complex is in an “open” inactive state, whereas H 2 O 2 induces allosteric rearrangement to the active “closed” conformation as a result of H 2 O 2 -dependent oxidative modification of cysteine residues (- SOH ), including S -glutathionylation (- SSG ). Such oxidative modification, followed by dissociation of AID from α-helix C and activation of AMPKα, can be achieved without binding of β/γ subunit. In the heterotrimeric AMPK complex, oxidative modification of the α and β subunits can also facilitate AMP-dependent activation of the kinase domain.
Figure Legend Snippet: Putative mechanism of AMPK activation by H 2 O 2 . The AMPKαβγ complex is in an “open” inactive state, whereas H 2 O 2 induces allosteric rearrangement to the active “closed” conformation as a result of H 2 O 2 -dependent oxidative modification of cysteine residues (- SOH ), including S -glutathionylation (- SSG ). Such oxidative modification, followed by dissociation of AID from α-helix C and activation of AMPKα, can be achieved without binding of β/γ subunit. In the heterotrimeric AMPK complex, oxidative modification of the α and β subunits can also facilitate AMP-dependent activation of the kinase domain.

Techniques Used: Activation Assay, Modification, Binding Assay

H 2 O 2 induces oxidation and activation of AMPK without depletion of cellular ATP. A–D , HEK 293 cells loaded with DCFH-DA were cultured with GO (10 milliunits/ml) for 0, 10, 20, or 40 min, and then images were acquired using confocal microscopy ( A ). B shows the ADP/ATP ratios in cells incubated with GO (10 milliunits/ml) for the indicated time periods, whereas C and D show representative Western blots of AMPK subunits and phospho-ACC ( pACC ) and mean ± S.D. ( error bars ) obtained from two experiments that utilized HEK 293 cells treated with GO for 0–50 min. E , HEK 293 cells were loaded with EE-GSH-biotin and then GO (10 milliunits/ml) included in culture medium for the indicated time period. Cell extracts were subjected to pull-down with streptavidin-agarose, followed by Western blotting with antibodies specific for AMPKα or phospho-Thr 172 -AMPK ( p-AMPK ) ( input , levels of AMPK or phospho-Thr 172 in cell extract prior to pull-down assay; Pull down , the amount of AMPK or phospho-Thr 172 -AMPK obtained after precipitation with streptavidin-agarose). Shown is the mean ± S.D. obtained from two experiments.
Figure Legend Snippet: H 2 O 2 induces oxidation and activation of AMPK without depletion of cellular ATP. A–D , HEK 293 cells loaded with DCFH-DA were cultured with GO (10 milliunits/ml) for 0, 10, 20, or 40 min, and then images were acquired using confocal microscopy ( A ). B shows the ADP/ATP ratios in cells incubated with GO (10 milliunits/ml) for the indicated time periods, whereas C and D show representative Western blots of AMPK subunits and phospho-ACC ( pACC ) and mean ± S.D. ( error bars ) obtained from two experiments that utilized HEK 293 cells treated with GO for 0–50 min. E , HEK 293 cells were loaded with EE-GSH-biotin and then GO (10 milliunits/ml) included in culture medium for the indicated time period. Cell extracts were subjected to pull-down with streptavidin-agarose, followed by Western blotting with antibodies specific for AMPKα or phospho-Thr 172 -AMPK ( p-AMPK ) ( input , levels of AMPK or phospho-Thr 172 in cell extract prior to pull-down assay; Pull down , the amount of AMPK or phospho-Thr 172 -AMPK obtained after precipitation with streptavidin-agarose). Shown is the mean ± S.D. obtained from two experiments.

Techniques Used: Activation Assay, Cell Culture, Confocal Microscopy, Incubation, Western Blot, Pull Down Assay

23) Product Images from "IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice, et al. IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice"

Article Title: IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice, et al. IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13610

Knock‐down of AMPKα induces macrophage autophagy. Purified PMΦs from normal mice were transfected with pooled siRNAs targeting α‐subunit of AMPK or negative control (NC) siRNA using Lipofectamine 2000 (Lipo 2000) as described in Methods. 48 h later, (A) total AMPKα and phosphorylated AMPKα were detected by Western blotting. Blots shown are representative of three independent experiments. B‐D, cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. B and C, LC3II expression was detected by FCM. B, FCM plots are representative of experiments. C, Data were MFI ± SD of three independent experiments. D, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ .05, ** P ≤ .01, *** P ≤ .001)
Figure Legend Snippet: Knock‐down of AMPKα induces macrophage autophagy. Purified PMΦs from normal mice were transfected with pooled siRNAs targeting α‐subunit of AMPK or negative control (NC) siRNA using Lipofectamine 2000 (Lipo 2000) as described in Methods. 48 h later, (A) total AMPKα and phosphorylated AMPKα were detected by Western blotting. Blots shown are representative of three independent experiments. B‐D, cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. B and C, LC3II expression was detected by FCM. B, FCM plots are representative of experiments. C, Data were MFI ± SD of three independent experiments. D, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ .05, ** P ≤ .01, *** P ≤ .001)

Techniques Used: Purification, Mouse Assay, Transfection, Negative Control, Western Blot, Expressing, Transmission Electron Microscopy

IL‐7 suppresses macrophage autophagy via AMP‐activated protein kinase (AMPK). Purified PMΦs from normal mice were treated as described in Figure 3 A legend. A and B, Phosphorylated AMPKα and total AMPKα were detected by Western blotting. Scanning densitometric analysis was performed, and the integrated optical density (IOD) of each band was normalized to corresponding β‐actin. A, Blots shown are representative of experiments. B, Data were means ± SD of three independent experiments. C‐H, Purified PMΦs from normal mice were pre‐treated with Met or compound C or an equal volume of solvent (PBS or DMSO) for 30 min; then cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. C, D, F and G, LC3II expression was detected by FCM. C and F, FCM plots are representative of experiments. D and G, Data were MFI ± SD of three independent experiments. E and H, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001)
Figure Legend Snippet: IL‐7 suppresses macrophage autophagy via AMP‐activated protein kinase (AMPK). Purified PMΦs from normal mice were treated as described in Figure 3 A legend. A and B, Phosphorylated AMPKα and total AMPKα were detected by Western blotting. Scanning densitometric analysis was performed, and the integrated optical density (IOD) of each band was normalized to corresponding β‐actin. A, Blots shown are representative of experiments. B, Data were means ± SD of three independent experiments. C‐H, Purified PMΦs from normal mice were pre‐treated with Met or compound C or an equal volume of solvent (PBS or DMSO) for 30 min; then cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. C, D, F and G, LC3II expression was detected by FCM. C and F, FCM plots are representative of experiments. D and G, Data were MFI ± SD of three independent experiments. E and H, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001)

Techniques Used: Purification, Mouse Assay, Western Blot, Expressing, Transmission Electron Microscopy

24) Product Images from "AMPK Regulates Circadian Rhythms in a Tissue- and Isoform-Specific Manner"

Article Title: AMPK Regulates Circadian Rhythms in a Tissue- and Isoform-Specific Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018450

Altered free-running period in AMPKα-deficient mice. (A–B) The activity rhythm was monitored by wheel running under light:dark cycles 12 hr:12 hr (LD) or under constant darkness (DD). Activity records of representative AMPKα1−/− and AMPKα2 −/− mice and their wild-type littermates are shown in double plotted actograms. Each horizontal line represents a 48 hr period and the vertical bars represent wheel running in 10-minute bins (n = 5–6). # P
Figure Legend Snippet: Altered free-running period in AMPKα-deficient mice. (A–B) The activity rhythm was monitored by wheel running under light:dark cycles 12 hr:12 hr (LD) or under constant darkness (DD). Activity records of representative AMPKα1−/− and AMPKα2 −/− mice and their wild-type littermates are shown in double plotted actograms. Each horizontal line represents a 48 hr period and the vertical bars represent wheel running in 10-minute bins (n = 5–6). # P

Techniques Used: Mouse Assay, Activity Assay

Circadian oscillation of AMPK activity in hypothalamus across the 24 hr light-dark cycle. Left, A representative Western blot showing the phosphorylation level of AMPK (T172) in extracts from mouse hypothalamus which were harvested at 4 hr intervals in a 12h light:12h dark (LD) cycle. The entire hypothalamus from 3 months old male C57BL/6J mice was used for this experiment. Right, Quantification of phosphorylated AMPKα (T172) is shown in arbitrary units (n = 3–4 per time point). Average AMPK activity (arbitrary unit) was calculated by densitometric quantification of phosphorylated proteins normalized to total proteins. Lights-on (6am; light) is indicated by a white bar and lights-off (6pm; dark) is indicated by a black bar. Results are means ± S.E. * P
Figure Legend Snippet: Circadian oscillation of AMPK activity in hypothalamus across the 24 hr light-dark cycle. Left, A representative Western blot showing the phosphorylation level of AMPK (T172) in extracts from mouse hypothalamus which were harvested at 4 hr intervals in a 12h light:12h dark (LD) cycle. The entire hypothalamus from 3 months old male C57BL/6J mice was used for this experiment. Right, Quantification of phosphorylated AMPKα (T172) is shown in arbitrary units (n = 3–4 per time point). Average AMPK activity (arbitrary unit) was calculated by densitometric quantification of phosphorylated proteins normalized to total proteins. Lights-on (6am; light) is indicated by a white bar and lights-off (6pm; dark) is indicated by a black bar. Results are means ± S.E. * P

Techniques Used: Activity Assay, Western Blot, Mouse Assay

Disruption of circadian physiology in AMPKα-deficient mice. (A,B) Core body temperature was measured by telemetry. AMPKα1−/−, AMPKα2−/− and WT mice were monitored in LD for 7 days followed by DD for 14 days. Representative data (mean ± SE) is LD day 7. Lights on is indicated by a white bar and lights off is indicated by a black bar. The same WT data is plotted in A and B. (C) Amplitude calculated from cosinor analysis of WT, AMPKα1−/− and AMPKα2−/− mice in LD, DD day 3, 7, 10. Results are expressed as mean ± S.E. * P
Figure Legend Snippet: Disruption of circadian physiology in AMPKα-deficient mice. (A,B) Core body temperature was measured by telemetry. AMPKα1−/−, AMPKα2−/− and WT mice were monitored in LD for 7 days followed by DD for 14 days. Representative data (mean ± SE) is LD day 7. Lights on is indicated by a white bar and lights off is indicated by a black bar. The same WT data is plotted in A and B. (C) Amplitude calculated from cosinor analysis of WT, AMPKα1−/− and AMPKα2−/− mice in LD, DD day 3, 7, 10. Results are expressed as mean ± S.E. * P

Techniques Used: Mouse Assay

Rhythmic expression of AMPK activity is cell autonomous. (A) WT mefs were synchronized by forskolin and harvested at the indicated time point. Phosphorylated AMPKα (T172) and phosphorylated-ACC (S79) were assessed by Western blot. Average AMPK and ACC activity (arbitrary unit) were calculated by densitometric quantification of phosphorylated proteins normalized to total proteins. Experiments were repeated at least three times. (B) Expression level (arbitrary units) of mPer2, Bmal1 and PGC-1α in WT and AMPKα1/α2 double knockout (AMPK KO) mefs after synchronization with forskolin. Results are means ± S.E. * P
Figure Legend Snippet: Rhythmic expression of AMPK activity is cell autonomous. (A) WT mefs were synchronized by forskolin and harvested at the indicated time point. Phosphorylated AMPKα (T172) and phosphorylated-ACC (S79) were assessed by Western blot. Average AMPK and ACC activity (arbitrary unit) were calculated by densitometric quantification of phosphorylated proteins normalized to total proteins. Experiments were repeated at least three times. (B) Expression level (arbitrary units) of mPer2, Bmal1 and PGC-1α in WT and AMPKα1/α2 double knockout (AMPK KO) mefs after synchronization with forskolin. Results are means ± S.E. * P

Techniques Used: Expressing, Activity Assay, Western Blot, Pyrolysis Gas Chromatography, Double Knockout

25) Product Images from "Calorie restriction protects against experimental abdominal aortic aneurysms in mice"

Article Title: Calorie restriction protects against experimental abdominal aortic aneurysms in mice

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151794

VSMC-SIRT1 is the main sensor of CR in aortas. (A) SIRT1, SIRT3, phosphorylated (p-AMPKα) and total AMPKα (t-AMPKα), and phosphorylated (p-mTOR) and total mTOR (t-mTOR) protein expression in aortas of Apoe −/− mice in the indicated group. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the AL-Con group values. n = 5 per group. G, GAPDH. (B) SIRT1 activity in aortas of Apoe −/− mice from the indicated group. n = 5 per group. (C) Representative images of IF staining of SIRT1, VSMCs (αSMA), and nuclei (Hoechst) in the suprarenal aortas of Apoe −/− mice for the indicated groups. Anti-SIRT1 and anti-αSMA antibodies were replaced by normal IgG as a negative control. A, adventitia; L, lumen. Bars, 50 µm. (D) SIRT1, SIRT3, phosphorylated and total AMPKα, and phosphorylated and total mTOR protein expression in VSMCs (isolated from the suprarenal abdominal aortas of WT mice) that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the values of the AL-Con group. Experiments were performed in triplicate. (E and F) Relative mRNA expression of genes encoding mitochondrial respiratory chain subunits (E) and genes involved in glucose and lipid metabolism (F) in WT VSMCs that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Experiments were performed in triplicate. All values are shown as the means ± SEM. *, P
Figure Legend Snippet: VSMC-SIRT1 is the main sensor of CR in aortas. (A) SIRT1, SIRT3, phosphorylated (p-AMPKα) and total AMPKα (t-AMPKα), and phosphorylated (p-mTOR) and total mTOR (t-mTOR) protein expression in aortas of Apoe −/− mice in the indicated group. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the AL-Con group values. n = 5 per group. G, GAPDH. (B) SIRT1 activity in aortas of Apoe −/− mice from the indicated group. n = 5 per group. (C) Representative images of IF staining of SIRT1, VSMCs (αSMA), and nuclei (Hoechst) in the suprarenal aortas of Apoe −/− mice for the indicated groups. Anti-SIRT1 and anti-αSMA antibodies were replaced by normal IgG as a negative control. A, adventitia; L, lumen. Bars, 50 µm. (D) SIRT1, SIRT3, phosphorylated and total AMPKα, and phosphorylated and total mTOR protein expression in VSMCs (isolated from the suprarenal abdominal aortas of WT mice) that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Protein expression was detected by Western blotting (left) and quantified by densitometry (right). Quantitative results are normalized to the values of the AL-Con group. Experiments were performed in triplicate. (E and F) Relative mRNA expression of genes encoding mitochondrial respiratory chain subunits (E) and genes involved in glucose and lipid metabolism (F) in WT VSMCs that were incubated with serum from AL-Con mice or CR-Con mice for 48 h. Experiments were performed in triplicate. All values are shown as the means ± SEM. *, P

Techniques Used: Expressing, Mouse Assay, Western Blot, Activity Assay, Staining, Negative Control, Isolation, Incubation

26) Product Images from "Glucagon-Like Peptide-1 Cleavage Product GLP-1 (9-36) Amide Enhances Hippocampal Long-term Synaptic Plasticity in Correlation with Suppression of Kv4.2 expression and eEF2 Phosphorylation"

Article Title: Glucagon-Like Peptide-1 Cleavage Product GLP-1 (9-36) Amide Enhances Hippocampal Long-term Synaptic Plasticity in Correlation with Suppression of Kv4.2 expression and eEF2 Phosphorylation

Journal: Hippocampus

doi: 10.1002/hipo.22795

GLP-1 (9-36) administration in vivo leads to suppression of eEF2 phosphorylation. (a) Western blotting on hippocampal slices from mice treated with GLP-1 (9-36) (500 ng/g/day) demonstrated significant reduced levels of eEF2 phosphorylation at Thr56; (b) Acute treatment of hippocampal slices with GLP-1 (9-36) did not alter eEF2 phosphorylation; (c) Phosphorylation of AMPKα was not affected by GLP-1 (9-36) in vivo treatment; (d) Acute treatment of hippocampal slices with GLP-1 (9-36) led to increase of AMPKα phosphorylation; (e); Levels of eEF1A was not changed by GLP-1 (9-36) treatment in vivo ; (f) Acute treatment of hippocampal slices with GLP-1 (9-36) did not alter eEF1A levels. n=6 for GLP-1 (9-36)-treated group and n= 4 for saline vehicle-treated group. (g) SUnSET experiments on hippocampal slices from mice treated with GLP-1 (9-36) did not reveal any significant change of newly synthesized proteins assessed by puromycin incorporation. n=12 for GLP-1 (9-36) in vivo group and n= 8 for saline vehicle-treated group and GLP-1 (9-36) in vitro group. * p
Figure Legend Snippet: GLP-1 (9-36) administration in vivo leads to suppression of eEF2 phosphorylation. (a) Western blotting on hippocampal slices from mice treated with GLP-1 (9-36) (500 ng/g/day) demonstrated significant reduced levels of eEF2 phosphorylation at Thr56; (b) Acute treatment of hippocampal slices with GLP-1 (9-36) did not alter eEF2 phosphorylation; (c) Phosphorylation of AMPKα was not affected by GLP-1 (9-36) in vivo treatment; (d) Acute treatment of hippocampal slices with GLP-1 (9-36) led to increase of AMPKα phosphorylation; (e); Levels of eEF1A was not changed by GLP-1 (9-36) treatment in vivo ; (f) Acute treatment of hippocampal slices with GLP-1 (9-36) did not alter eEF1A levels. n=6 for GLP-1 (9-36)-treated group and n= 4 for saline vehicle-treated group. (g) SUnSET experiments on hippocampal slices from mice treated with GLP-1 (9-36) did not reveal any significant change of newly synthesized proteins assessed by puromycin incorporation. n=12 for GLP-1 (9-36) in vivo group and n= 8 for saline vehicle-treated group and GLP-1 (9-36) in vitro group. * p

Techniques Used: In Vivo, Western Blot, Mouse Assay, Synthesized, In Vitro

27) Product Images from "Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK) *"

Article Title: Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.646992

AMPK mediates the anti-inflammatory effects of palmitoleate. A and B , BMDM were treated with BSA alone, 0.5 m m PA, 0.5 m m PO, or 0.5 m m PA + 0.5 m m PO for 30 min, AMPKα phosphorylation was determined by immunoblotting ( A ), and cytosolic ATP concentration
Figure Legend Snippet: AMPK mediates the anti-inflammatory effects of palmitoleate. A and B , BMDM were treated with BSA alone, 0.5 m m PA, 0.5 m m PO, or 0.5 m m PA + 0.5 m m PO for 30 min, AMPKα phosphorylation was determined by immunoblotting ( A ), and cytosolic ATP concentration

Techniques Used: Concentration Assay

28) Product Images from "Semen Cuscutae Administration Improves Hepatic Lipid Metabolism and Adiposity in High Fat Diet-Induced Obese Mice"

Article Title: Semen Cuscutae Administration Improves Hepatic Lipid Metabolism and Adiposity in High Fat Diet-Induced Obese Mice

Journal: Nutrients

doi: 10.3390/nu11123035

Effect of the SC on the activation of hepatic AMPKα in vivo. Protein levels in liver tissue with p -AMPKα and AMPKα levels showing a representative western blot image. The results are expressed as mean ± S.E. of mice, and the same letter indicates no significant difference between two groups ( p
Figure Legend Snippet: Effect of the SC on the activation of hepatic AMPKα in vivo. Protein levels in liver tissue with p -AMPKα and AMPKα levels showing a representative western blot image. The results are expressed as mean ± S.E. of mice, and the same letter indicates no significant difference between two groups ( p

Techniques Used: Activation Assay, In Vivo, Western Blot, Mouse Assay

29) Product Images from "IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice, et al. IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice"

Article Title: IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice, et al. IL‐7 suppresses macrophage autophagy and promotes liver pathology in Schistosoma japonicum‐infected mice

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13610

Knock‐down of AMPKα induces macrophage autophagy. Purified PMΦs from normal mice were transfected with pooled siRNAs targeting α‐subunit of AMPK or negative control (NC) siRNA using Lipofectamine 2000 (Lipo 2000) as described in Methods. 48 h later, (A) total AMPKα and phosphorylated AMPKα were detected by Western blotting. Blots shown are representative of three independent experiments. B‐D, cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. B and C, LC3II expression was detected by FCM. B, FCM plots are representative of experiments. C, Data were MFI ± SD of three independent experiments. D, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ .05, ** P ≤ .01, *** P ≤ .001)
Figure Legend Snippet: Knock‐down of AMPKα induces macrophage autophagy. Purified PMΦs from normal mice were transfected with pooled siRNAs targeting α‐subunit of AMPK or negative control (NC) siRNA using Lipofectamine 2000 (Lipo 2000) as described in Methods. 48 h later, (A) total AMPKα and phosphorylated AMPKα were detected by Western blotting. Blots shown are representative of three independent experiments. B‐D, cells were treated with PBS, SEA, IL‐7 or SEA plus IL‐7 for another 24 h. B and C, LC3II expression was detected by FCM. B, FCM plots are representative of experiments. C, Data were MFI ± SD of three independent experiments. D, Autophagosomes were detected by TEM. Data were means ± SD of 150 macrophages from three independent experiments. (* P ≤ .05, ** P ≤ .01, *** P ≤ .001)

Techniques Used: Purification, Mouse Assay, Transfection, Negative Control, Western Blot, Expressing, Transmission Electron Microscopy

30) Product Images from "Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma"

Article Title: Citrate Synthase Expression Affects Tumor Phenotype and Drug Resistance in Human Ovarian Carcinoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115708

CS silencing affects AMPK/P38 MAPK pathway in ovarian cancer cell lines. ( A ) mRNA and (B) protein expression level of CS by real-time PCR and western blot in SKOV3 and A2780 cells after CS siRNA (100 nM) for 24 h and 48 h after transfection, respectively. ( C ) Decreased CS activity after 48 h transfection in SKOV3 and A2780 cells. ( D ) ATP level was examined 48 h after CS silencing. ( E, F ) p-AMPKα and p-p38 were analyzed in CS -silenced cancer cells by western blot. Mean ± SEM. * P
Figure Legend Snippet: CS silencing affects AMPK/P38 MAPK pathway in ovarian cancer cell lines. ( A ) mRNA and (B) protein expression level of CS by real-time PCR and western blot in SKOV3 and A2780 cells after CS siRNA (100 nM) for 24 h and 48 h after transfection, respectively. ( C ) Decreased CS activity after 48 h transfection in SKOV3 and A2780 cells. ( D ) ATP level was examined 48 h after CS silencing. ( E, F ) p-AMPKα and p-p38 were analyzed in CS -silenced cancer cells by western blot. Mean ± SEM. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Activity Assay

31) Product Images from "Cucurbitacin B and SCH772984 exhibit synergistic anti-pancreatic cancer activities by suppressing EGFR, PI3K/Akt/mTOR, STAT3 and ERK signaling"

Article Title: Cucurbitacin B and SCH772984 exhibit synergistic anti-pancreatic cancer activities by suppressing EGFR, PI3K/Akt/mTOR, STAT3 and ERK signaling

Journal: Oncotarget

doi: 10.18632/oncotarget.21704

CuB enhances ERK activity via AMPK activation ( A ) BxPC-3 and HPAC cells were treated with vehicle control or CuB for 24 h. Whole cell lysates were analyzed by Western blotting and probed with anti-AMPKα, -pAMPKα, -ERK, -pERK or -β-actin antibodies. ( B ) BxPC-3 and HPAC cells were treated with 0.3 µM CuB for up to 24 h. Cells were harvested and lysed. Protein extracts were analyzed by Western blotting and probed with the indicated antibodies. ( C ) BxPC-3 and HPAC cells were treated with CuB and compound C (C.C) alone or in combination for 24 h. Whole cell lysates were analyzed by Western blotting and probed with anti-AMPKα, -pAMPKα, -ERK, -pERK, -pS6 or -β-actin antibodies. ( D ) BxPC-3 and HPAC cells were infected with non-template control (NTC) or AMPKα CRISPR lentivirus (#gRNA). Cells were then treated with or without CuB for 24 h. Whole cell lysates were analyzed by Western blotting and probed with the indicated antibodies. Experiments were performed at least 3 independent times, and representative Western blots are shown.
Figure Legend Snippet: CuB enhances ERK activity via AMPK activation ( A ) BxPC-3 and HPAC cells were treated with vehicle control or CuB for 24 h. Whole cell lysates were analyzed by Western blotting and probed with anti-AMPKα, -pAMPKα, -ERK, -pERK or -β-actin antibodies. ( B ) BxPC-3 and HPAC cells were treated with 0.3 µM CuB for up to 24 h. Cells were harvested and lysed. Protein extracts were analyzed by Western blotting and probed with the indicated antibodies. ( C ) BxPC-3 and HPAC cells were treated with CuB and compound C (C.C) alone or in combination for 24 h. Whole cell lysates were analyzed by Western blotting and probed with anti-AMPKα, -pAMPKα, -ERK, -pERK, -pS6 or -β-actin antibodies. ( D ) BxPC-3 and HPAC cells were infected with non-template control (NTC) or AMPKα CRISPR lentivirus (#gRNA). Cells were then treated with or without CuB for 24 h. Whole cell lysates were analyzed by Western blotting and probed with the indicated antibodies. Experiments were performed at least 3 independent times, and representative Western blots are shown.

Techniques Used: Activity Assay, Activation Assay, Western Blot, Infection, CRISPR

32) Product Images from "Fenofibrate Suppresses Oral Tumorigenesis via Reprogramming Metabolic Processes: Potential Drug Repurposing for Oral Cancer"

Article Title: Fenofibrate Suppresses Oral Tumorigenesis via Reprogramming Metabolic Processes: Potential Drug Repurposing for Oral Cancer

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.13851

Dose-dependent changes in AMPK/mTOR or Akt/mTOR signaling protein levels in SAS cells treated with fenofibrate. SAS cells were treated with 0, 12.5, 25, 50, and 75 μM fenofibrate for 4 hrs. Changes in the expression levels of AMPKα, p-AMPKα (Thr172), AMPKβ1/2, p-AMPKβ1 (Ser108), AMPKγ1, Akt1+2+3, p-Akt1(Ser473), tuberous sclerosis complex-1 (TSC1), TSC2, Ras homolog enriched in brain (Rheb), raptor, p-raptor (Ser792), PRAS40, and p-PRAS40 (Thr246) were measured by Western blot analysis. β-Actin was used as an internal control.
Figure Legend Snippet: Dose-dependent changes in AMPK/mTOR or Akt/mTOR signaling protein levels in SAS cells treated with fenofibrate. SAS cells were treated with 0, 12.5, 25, 50, and 75 μM fenofibrate for 4 hrs. Changes in the expression levels of AMPKα, p-AMPKα (Thr172), AMPKβ1/2, p-AMPKβ1 (Ser108), AMPKγ1, Akt1+2+3, p-Akt1(Ser473), tuberous sclerosis complex-1 (TSC1), TSC2, Ras homolog enriched in brain (Rheb), raptor, p-raptor (Ser792), PRAS40, and p-PRAS40 (Thr246) were measured by Western blot analysis. β-Actin was used as an internal control.

Techniques Used: Expressing, Western Blot

33) Product Images from "Flavonoids of Polygonum hydropiper L. attenuates lipopolysaccharide-induced inflammatory injury via suppressing phosphorylation in MAPKs pathways"

Article Title: Flavonoids of Polygonum hydropiper L. attenuates lipopolysaccharide-induced inflammatory injury via suppressing phosphorylation in MAPKs pathways

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-016-1001-8

Effects of FNP on phosphorylation of MAPKs and AMPK in LPS-stimulated RAW264.7 cells. Notes: The cells were pretreated with different concentrations (40 μg/mL and 80 μg/mL) of FNP for 2 h or 4 h and stimulated with 1 μg/ml of LPS for 1 h. Total cellular proteins (50 μg) were separated by SDS-PAGE, then transferred to PVDF membrane and detected by Western blot analysis. Western blot results were shown in ( a ). Quantification of p-AMPKα, p-ERK1/2, p-P38, p-JNK and p-c-JUN protein expression was normalized to AMPKα, ERK1/2, P38, JNK and c-JUN using a densitometer. Each column represented as the means ± SD from three independent experiments. Results of 4h pretreatment were shown in ( b ) and results of 2 h pretreatment were shown in ( c ). # P
Figure Legend Snippet: Effects of FNP on phosphorylation of MAPKs and AMPK in LPS-stimulated RAW264.7 cells. Notes: The cells were pretreated with different concentrations (40 μg/mL and 80 μg/mL) of FNP for 2 h or 4 h and stimulated with 1 μg/ml of LPS for 1 h. Total cellular proteins (50 μg) were separated by SDS-PAGE, then transferred to PVDF membrane and detected by Western blot analysis. Western blot results were shown in ( a ). Quantification of p-AMPKα, p-ERK1/2, p-P38, p-JNK and p-c-JUN protein expression was normalized to AMPKα, ERK1/2, P38, JNK and c-JUN using a densitometer. Each column represented as the means ± SD from three independent experiments. Results of 4h pretreatment were shown in ( b ) and results of 2 h pretreatment were shown in ( c ). # P

Techniques Used: SDS Page, Western Blot, Expressing

34) Product Images from "AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation"

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

Journal: Oncotarget

doi: 10.18632/oncotarget.7404

Disruption of AMPKα1(S173) increases cell death during glucose starvation in hepatocarcinoma derived cells C3A cells stably expressing either AMPKα1(S173C) (S173C), AMPKα1(S173D) (S173D) or wild type AMPKα1(S173) (WT) were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. After 12 h of glucose deprivation cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172), AMPKα and Puma. α Tubulin was used as loading control. The blots in the picture are representative of 3 independent experiments. B. Cells attached to microplates were cultured for 0, 24, 48 and 72 h. MTT assay was performed as described in Materials and Methods . Results are presented as the percentage of the absorbance of the cells at 0 h. C. After 36 h cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent fold increase respect to WT controls (WT C) in the proportion of cells undergoing apoptosis (Annexin V positive). WT C apoptotic cells (mean value): 9%. Values represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: Disruption of AMPKα1(S173) increases cell death during glucose starvation in hepatocarcinoma derived cells C3A cells stably expressing either AMPKα1(S173C) (S173C), AMPKα1(S173D) (S173D) or wild type AMPKα1(S173) (WT) were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. After 12 h of glucose deprivation cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172), AMPKα and Puma. α Tubulin was used as loading control. The blots in the picture are representative of 3 independent experiments. B. Cells attached to microplates were cultured for 0, 24, 48 and 72 h. MTT assay was performed as described in Materials and Methods . Results are presented as the percentage of the absorbance of the cells at 0 h. C. After 36 h cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent fold increase respect to WT controls (WT C) in the proportion of cells undergoing apoptosis (Annexin V positive). WT C apoptotic cells (mean value): 9%. Values represent the mean ± SEM of 3 experiments. * P

Techniques Used: Derivative Assay, Stable Transfection, Expressing, Incubation, Western Blot, Cell Culture, MTT Assay, Staining, Flow Cytometry, Cytometry

AMPK and PKA activation during glucose restriction C3A cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). Cell lysates were obtained and analyzed by western blotting. A. Protein levels of P-AMPKα(T172) and AMPKα were detected at 2 and 6 h of glucose deprivation. B. Phosphorylated PKA substrates (RXXS/T-P residues) were detected after 2 h of treatments. Cells incubated with 100 μM dbcAMP (dbcAMP) were used as positive control of PKA phosphorylation. Bars represent band densities of PKA substrates weighted 37, 80 and 140 kDa. α Tubulin was used as loading control. Values represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: AMPK and PKA activation during glucose restriction C3A cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). Cell lysates were obtained and analyzed by western blotting. A. Protein levels of P-AMPKα(T172) and AMPKα were detected at 2 and 6 h of glucose deprivation. B. Phosphorylated PKA substrates (RXXS/T-P residues) were detected after 2 h of treatments. Cells incubated with 100 μM dbcAMP (dbcAMP) were used as positive control of PKA phosphorylation. Bars represent band densities of PKA substrates weighted 37, 80 and 140 kDa. α Tubulin was used as loading control. Values represent the mean ± SEM of 3 experiments. * P

Techniques Used: Activation Assay, Incubation, Western Blot, Positive Control

Effects of AMPK knock down on cell cycle progression and cell death during glucose deprivation in liver cancer cells C3A (A, B, C) HuH-7 and SK-Hep-1 (D) cells were transfected with a siRNA specifically targeted to AMPKα1 mRNA (AMPK KD), or with a siRNA control (Control), and allowed to grow for 48 hours. A. AMPKα expression was analyzed by western blot, in Control and AMPK KD cells. α Tubulin was used as loading control. In panels B, C and D control and AMPK KD cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0) during 24 h. B. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The figure shows typical outputs of the cytometric analysis and percentages of C3A cells in the different phases of the cell cycle, an experiment representative of 3 independent experiments. C. C3A cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent increase in the percentages of cells undergoing apoptosis (Annexin V positive) relative to controls (C). Control apoptotic cells (mean value): 8%. Values represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: Effects of AMPK knock down on cell cycle progression and cell death during glucose deprivation in liver cancer cells C3A (A, B, C) HuH-7 and SK-Hep-1 (D) cells were transfected with a siRNA specifically targeted to AMPKα1 mRNA (AMPK KD), or with a siRNA control (Control), and allowed to grow for 48 hours. A. AMPKα expression was analyzed by western blot, in Control and AMPK KD cells. α Tubulin was used as loading control. In panels B, C and D control and AMPK KD cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0) during 24 h. B. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The figure shows typical outputs of the cytometric analysis and percentages of C3A cells in the different phases of the cell cycle, an experiment representative of 3 independent experiments. C. C3A cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent increase in the percentages of cells undergoing apoptosis (Annexin V positive) relative to controls (C). Control apoptotic cells (mean value): 8%. Values represent the mean ± SEM of 3 experiments. * P

Techniques Used: Transfection, Expressing, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry

Regulation of AMPK activation by PKA phosphorylation in hepatic cancer cells C3A cells were incubated for 36 h (A) and 24 h (B) with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. C and Glc0 cells were incubated in the absence or presence of 100 μM dbcAMP (dbcAMP) or/and 1 mM AICAR (AICAR). Cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172) and AMPKα. β Actin was used as loading control. Bars represent the mean ± SEM of 3 experiments. * P
Figure Legend Snippet: Regulation of AMPK activation by PKA phosphorylation in hepatic cancer cells C3A cells were incubated for 36 h (A) and 24 h (B) with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. C and Glc0 cells were incubated in the absence or presence of 100 μM dbcAMP (dbcAMP) or/and 1 mM AICAR (AICAR). Cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172) and AMPKα. β Actin was used as loading control. Bars represent the mean ± SEM of 3 experiments. * P

Techniques Used: Activation Assay, Incubation, Western Blot

35) Product Images from "Cyclic Stretch Negatively Regulates IL-1β Secretion Through the Inhibition of NLRP3 Inflammasome Activation by Attenuating the AMP Kinase Pathway"

Article Title: Cyclic Stretch Negatively Regulates IL-1β Secretion Through the Inhibition of NLRP3 Inflammasome Activation by Attenuating the AMP Kinase Pathway

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.00802

Cyclic stretch inhibits NLRP3 inflammasome activation and is partially dependent on the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. J774.1 cells were primed with 100 ng/ml LPS for 4 h, treated with 20 μM of compound C (C.C.) for 30 min, and then stimulated with 1 mM ATP for 2 h in the continuous presence of LPS and compound C (A–C) . All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the culture medium during the cell culture. (A) : The amounts of IL-1β in the supernatant were analyzed by ELISA. (B) : LDH activity in supernatants was measured by a LDH assay kit at 490 nm. (C) : Cells were labeled with a FLICA probe conjugated with FAM with the indication of green fluorescence and nuclei were visualized by staining with Hoechst 33342 (blue) (magnification: × 200; scale bars are 50 μm). (D,E) : J774.1 cells were exposed to CS of 20% elongation at a frequency of 10 cycles/min for the first 2 h during a treatment with 100 ng/ml LPS for 4 h, followed by a stimulation with ATP for 2 h in the continuous presence of LPS. (D) : Cell lysates were analyzed by Western blotting with phosphorylated-AMPKα and AMPKα antibodies. An antibody against β-actin was used as a control (molecular mass, phosphorylated-AMPKα and AMPKα: 62 kDa). (E) : The expression level of phosphorylated-AMPKα was quantified via densitometry scanning. Relative expression levels of phosphorylated-AMPKα were normalized to AMPKα. Representative results of three independent experiments are shown. Significance is indicated ( ∗ P
Figure Legend Snippet: Cyclic stretch inhibits NLRP3 inflammasome activation and is partially dependent on the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. J774.1 cells were primed with 100 ng/ml LPS for 4 h, treated with 20 μM of compound C (C.C.) for 30 min, and then stimulated with 1 mM ATP for 2 h in the continuous presence of LPS and compound C (A–C) . All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the culture medium during the cell culture. (A) : The amounts of IL-1β in the supernatant were analyzed by ELISA. (B) : LDH activity in supernatants was measured by a LDH assay kit at 490 nm. (C) : Cells were labeled with a FLICA probe conjugated with FAM with the indication of green fluorescence and nuclei were visualized by staining with Hoechst 33342 (blue) (magnification: × 200; scale bars are 50 μm). (D,E) : J774.1 cells were exposed to CS of 20% elongation at a frequency of 10 cycles/min for the first 2 h during a treatment with 100 ng/ml LPS for 4 h, followed by a stimulation with ATP for 2 h in the continuous presence of LPS. (D) : Cell lysates were analyzed by Western blotting with phosphorylated-AMPKα and AMPKα antibodies. An antibody against β-actin was used as a control (molecular mass, phosphorylated-AMPKα and AMPKα: 62 kDa). (E) : The expression level of phosphorylated-AMPKα was quantified via densitometry scanning. Relative expression levels of phosphorylated-AMPKα were normalized to AMPKα. Representative results of three independent experiments are shown. Significance is indicated ( ∗ P

Techniques Used: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay, Labeling, Fluorescence, Staining, Western Blot, Expressing

36) Product Images from "Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1"

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.07.030

Metformin increases CTL activity through the AMPK/PD-L1 axis. (A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm 2 in (D) and 262,144 μm 2 in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. * P , 0.01~0.05, ** P , 0.001~0.01, # P ,
Figure Legend Snippet: Metformin increases CTL activity through the AMPK/PD-L1 axis. (A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm 2 in (D) and 262,144 μm 2 in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. * P , 0.01~0.05, ** P , 0.001~0.01, # P ,

Techniques Used: CTL Assay, Activity Assay, Injection, Mouse Assay, Imaging, Immunostaining, Staining, Marker, Multiple Displacement Amplification, Cell Culture, Expressing, Flow Cytometry, Quantitation Assay, Binding Assay, Labeling

The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant (A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm 2 . (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. * P , 0.01~0.05, ** P , 0.001~0.01, and # P ,
Figure Legend Snippet: The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant (A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm 2 . (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. * P , 0.01~0.05, ** P , 0.001~0.01, and # P ,

Techniques Used: Injection, Mouse Assay, Immunostaining, Staining, Immunohistochemistry, Expressing, Western Blot

37) Product Images from "Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1"

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.07.030

Metformin increases CTL activity through the AMPK/PD-L1 axis. (A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm 2 in (D) and 262,144 μm 2 in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. * P , 0.01~0.05, ** P , 0.001~0.01, # P ,
Figure Legend Snippet: Metformin increases CTL activity through the AMPK/PD-L1 axis. (A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm 2 in (D) and 262,144 μm 2 in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. * P , 0.01~0.05, ** P , 0.001~0.01, # P ,

Techniques Used: CTL Assay, Activity Assay, Injection, Mouse Assay, Imaging, Immunostaining, Staining, Marker, Multiple Displacement Amplification, Cell Culture, Expressing, Flow Cytometry, Quantitation Assay, Binding Assay, Labeling

The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant (A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm 2 . (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. * P , 0.01~0.05, ** P , 0.001~0.01, and # P ,
Figure Legend Snippet: The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant (A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm 2 . (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. * P , 0.01~0.05, ** P , 0.001~0.01, and # P ,

Techniques Used: Injection, Mouse Assay, Immunostaining, Staining, Immunohistochemistry, Expressing, Western Blot

38) Product Images from "Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation"

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2018.00098

Knockdown of AMPK by siRNA attenuated BA-mediated microglia polarization in BV-2 microglial cells. Cells were first transfected with 40 nM AMPKα siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of AMPKα protein. (B,C) AMPKα knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) AMPKα knockdown attenuated BA-enhanced IL-10 release and mRNA expression of CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P
Figure Legend Snippet: Knockdown of AMPK by siRNA attenuated BA-mediated microglia polarization in BV-2 microglial cells. Cells were first transfected with 40 nM AMPKα siRNA for 24 h, and then treated with BA for 1 h, followed by exposure to LPS stimulation for 6 or 24 h. (A) Transfection for 24 h was able to decrease the expression of AMPKα protein. (B,C) AMPKα knockdown decreased BA-mediated inhibition of TNF-α production, and mRNA expression of TNF-α and iNOS in LPS-stimulated BV-2 cells. (D–F) AMPKα knockdown attenuated BA-enhanced IL-10 release and mRNA expression of CD206 and Arg-1. Data are presented as means ± SEM of three independent experiments in triplicate. Control group was that treated with control siRNA but not BA or LPS. Two columns sharing the same letter are significantly different ( P

Techniques Used: Transfection, Expressing, Inhibition

39) Product Images from "Fumagillin Prodrug Nanotherapy Suppresses Macrophage Inflammatory Response via Endothelial Nitric Oxide"

Article Title: Fumagillin Prodrug Nanotherapy Suppresses Macrophage Inflammatory Response via Endothelial Nitric Oxide

Journal: ACS Nano

doi: 10.1021/nn502372n

Fum-PD-induced NO indirectly suppresses MΦ inflammatory response in vitro . (A) Fum-PD had no direct effect on the release of TNF-α from MΦ. The experiment was repeated three times with similar results. (B, C) Fum-PD dose-dependently suppressed TNF-α release from EC–MΦ cocultures. The effect was reversed by l -NAME (20 μM), but not the inactive isomer d -NAME (20 μM). Values represent mean ± SEM of triplicate samples derived from three independent experiments. (D) EC–MΦ cocultures were exposed to Fum-PD (0–40 nM) or SNAP (25 μg/mL) without or with l -NAME (0–20 μM) and harvested at 48 h, and cell lysates were probed for phospho (P)-AMPKα, AMPKα, phospho (P)-p65, and p65. (E) EC–MΦ cocultures were exposed to Fum-PD (20 nM) without or with compound C (CC) at the indicated concentration. Supernatants were harvested and analyzed for TNF-α. The experiment is representative of two independent experiments with similar results. (F) EC–MΦ cocultures were exposed to Fum-PD without or with l -NAME (20 μM), and 48 h supernatants were assayed for IL-10. (G) RNA obtained from cocultures was analyzed by real-time PCR for expression of arginase 1, an M2 marker. * p
Figure Legend Snippet: Fum-PD-induced NO indirectly suppresses MΦ inflammatory response in vitro . (A) Fum-PD had no direct effect on the release of TNF-α from MΦ. The experiment was repeated three times with similar results. (B, C) Fum-PD dose-dependently suppressed TNF-α release from EC–MΦ cocultures. The effect was reversed by l -NAME (20 μM), but not the inactive isomer d -NAME (20 μM). Values represent mean ± SEM of triplicate samples derived from three independent experiments. (D) EC–MΦ cocultures were exposed to Fum-PD (0–40 nM) or SNAP (25 μg/mL) without or with l -NAME (0–20 μM) and harvested at 48 h, and cell lysates were probed for phospho (P)-AMPKα, AMPKα, phospho (P)-p65, and p65. (E) EC–MΦ cocultures were exposed to Fum-PD (20 nM) without or with compound C (CC) at the indicated concentration. Supernatants were harvested and analyzed for TNF-α. The experiment is representative of two independent experiments with similar results. (F) EC–MΦ cocultures were exposed to Fum-PD without or with l -NAME (20 μM), and 48 h supernatants were assayed for IL-10. (G) RNA obtained from cocultures was analyzed by real-time PCR for expression of arginase 1, an M2 marker. * p

Techniques Used: In Vitro, Derivative Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing, Marker

40) Product Images from "Resveratrol enhances brown adipocyte formation and function by activating AMP-activated protein kinase (AMPK) α1 in mice fed high-fat diet"

Article Title: Resveratrol enhances brown adipocyte formation and function by activating AMP-activated protein kinase (AMPK) α1 in mice fed high-fat diet

Journal: Molecular nutrition & food research

doi: 10.1002/mnfr.201600746

Dietary supplementation of resveratrol enhanced iBAT formation and function in mice fed HFD. A) Representative images of H E and UCP1 IHC staining in sections of iBAT of control and 0.1% Resv treated mice (scale bar, 100 µm). Arrow indicates the large lipid droplets (diameter ~35 µm) in control group. B) The number of nuclei in microscopic fields selected at random from H E stained sections were compared between control and 0.1% Resv groups. C) Western blot analyses of brown adipogenic marker gene (PRDM16), thermogenic genes (UCP-1, Cyto C), p-AMPKα and t-AMPKα, and β-actin was used as the loading control. D) Mean ± SEM of immunoblotting bands and the intensities of the bands were expressed as arbitrary units. *P
Figure Legend Snippet: Dietary supplementation of resveratrol enhanced iBAT formation and function in mice fed HFD. A) Representative images of H E and UCP1 IHC staining in sections of iBAT of control and 0.1% Resv treated mice (scale bar, 100 µm). Arrow indicates the large lipid droplets (diameter ~35 µm) in control group. B) The number of nuclei in microscopic fields selected at random from H E stained sections were compared between control and 0.1% Resv groups. C) Western blot analyses of brown adipogenic marker gene (PRDM16), thermogenic genes (UCP-1, Cyto C), p-AMPKα and t-AMPKα, and β-actin was used as the loading control. D) Mean ± SEM of immunoblotting bands and the intensities of the bands were expressed as arbitrary units. *P

Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Western Blot, Marker

Resveratrol activated AMPKα in differentiated iBAT SVCs separated from the wild-type but not AMPKα1 conditional knockout mice. A) Western blot analysis of p-AMPKα, t-AMPKα of differentiated iBAT SVCs. β-actin was used as loading control. B) Mean ± SEM of immunoblotting bands and the intensities of the bands were expressed as arbitrary units. *P
Figure Legend Snippet: Resveratrol activated AMPKα in differentiated iBAT SVCs separated from the wild-type but not AMPKα1 conditional knockout mice. A) Western blot analysis of p-AMPKα, t-AMPKα of differentiated iBAT SVCs. β-actin was used as loading control. B) Mean ± SEM of immunoblotting bands and the intensities of the bands were expressed as arbitrary units. *P

Techniques Used: Knock-Out, Mouse Assay, Western Blot

41) Product Images from "Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression"

Article Title: Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2016.00445

Median (± SEM) [ n = 10 (AL 10% PRO; ), n = 10 (AL 32% PRO; ), n = 10 (CR 10% PRO; ), n = 10 (CR 32% PRO; □)] mRNA (A) and mean (± SEM) total AMPKα and PGC-1α (B) expression and citrate synthase activity (C) . Data analyzed using a univariate ANOVA with Bonferroni correction to determine main effects of energy (AL vs. CR), protein (10 vs. 32%) and energy-by-protein interactions. * CR different from AL; P
Figure Legend Snippet: Median (± SEM) [ n = 10 (AL 10% PRO; ), n = 10 (AL 32% PRO; ), n = 10 (CR 10% PRO; ), n = 10 (CR 32% PRO; □)] mRNA (A) and mean (± SEM) total AMPKα and PGC-1α (B) expression and citrate synthase activity (C) . Data analyzed using a univariate ANOVA with Bonferroni correction to determine main effects of energy (AL vs. CR), protein (10 vs. 32%) and energy-by-protein interactions. * CR different from AL; P

Techniques Used: Pyrolysis Gas Chromatography, Expressing, Activity Assay

42) Product Images from "Cyclic Stretch Negatively Regulates IL-1β Secretion Through the Inhibition of NLRP3 Inflammasome Activation by Attenuating the AMP Kinase Pathway"

Article Title: Cyclic Stretch Negatively Regulates IL-1β Secretion Through the Inhibition of NLRP3 Inflammasome Activation by Attenuating the AMP Kinase Pathway

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.00802

Cyclic stretch inhibits NLRP3 inflammasome activation and is partially dependent on the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. J774.1 cells were primed with 100 ng/ml LPS for 4 h, treated with 20 μM of compound C (C.C.) for 30 min, and then stimulated with 1 mM ATP for 2 h in the continuous presence of LPS and compound C (A–C) . All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the culture medium during the cell culture. (A) : The amounts of IL-1β in the supernatant were analyzed by ELISA. (B) : LDH activity in supernatants was measured by a LDH assay kit at 490 nm. (C) : Cells were labeled with a FLICA probe conjugated with FAM with the indication of green fluorescence and nuclei were visualized by staining with Hoechst 33342 (blue) (magnification: × 200; scale bars are 50 μm). (D,E) : J774.1 cells were exposed to CS of 20% elongation at a frequency of 10 cycles/min for the first 2 h during a treatment with 100 ng/ml LPS for 4 h, followed by a stimulation with ATP for 2 h in the continuous presence of LPS. (D) : Cell lysates were analyzed by Western blotting with phosphorylated-AMPKα and AMPKα antibodies. An antibody against β-actin was used as a control (molecular mass, phosphorylated-AMPKα and AMPKα: 62 kDa). (E) : The expression level of phosphorylated-AMPKα was quantified via densitometry scanning. Relative expression levels of phosphorylated-AMPKα were normalized to AMPKα. Representative results of three independent experiments are shown. Significance is indicated ( ∗ P
Figure Legend Snippet: Cyclic stretch inhibits NLRP3 inflammasome activation and is partially dependent on the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. J774.1 cells were primed with 100 ng/ml LPS for 4 h, treated with 20 μM of compound C (C.C.) for 30 min, and then stimulated with 1 mM ATP for 2 h in the continuous presence of LPS and compound C (A–C) . All samples, including the control, were adjusted to contain 0.1% (v/v) DMSO in the culture medium during the cell culture. (A) : The amounts of IL-1β in the supernatant were analyzed by ELISA. (B) : LDH activity in supernatants was measured by a LDH assay kit at 490 nm. (C) : Cells were labeled with a FLICA probe conjugated with FAM with the indication of green fluorescence and nuclei were visualized by staining with Hoechst 33342 (blue) (magnification: × 200; scale bars are 50 μm). (D,E) : J774.1 cells were exposed to CS of 20% elongation at a frequency of 10 cycles/min for the first 2 h during a treatment with 100 ng/ml LPS for 4 h, followed by a stimulation with ATP for 2 h in the continuous presence of LPS. (D) : Cell lysates were analyzed by Western blotting with phosphorylated-AMPKα and AMPKα antibodies. An antibody against β-actin was used as a control (molecular mass, phosphorylated-AMPKα and AMPKα: 62 kDa). (E) : The expression level of phosphorylated-AMPKα was quantified via densitometry scanning. Relative expression levels of phosphorylated-AMPKα were normalized to AMPKα. Representative results of three independent experiments are shown. Significance is indicated ( ∗ P

Techniques Used: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay, Labeling, Fluorescence, Staining, Western Blot, Expressing

43) Product Images from "Low Concentrations of Metformin Selectively Inhibit CD133+ Cell Proliferation in Pancreatic Cancer and Have Anticancer Action"

Article Title: Low Concentrations of Metformin Selectively Inhibit CD133+ Cell Proliferation in Pancreatic Cancer and Have Anticancer Action

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063969

Low concentrations of metformin inhibited Erk and mTOR phosphorylation and increased Akt phosphorylation of CD133 + pancreatic cancer cells. Pancreatic cancer cells were treated with metformin for 4 h (0.2 mM for AsPC-1, 0.1 mM for SW1990) and CD133 + cells were sorted by flow cytometry. Expression of AMPKα, Akt, Erk1/2, and mTOR and phosphorylation of CD133 + cells were evaluated by western blotting and the results were quantified using ImageJ V.1.46r (National Institutes of Health). Significant decreases of phospho-ERK1/2 and phospho-mTOR expression and a significant increase of phospho-Akt expression were observed in the metformin treated cells. Error bars represent the standard deviation. * P
Figure Legend Snippet: Low concentrations of metformin inhibited Erk and mTOR phosphorylation and increased Akt phosphorylation of CD133 + pancreatic cancer cells. Pancreatic cancer cells were treated with metformin for 4 h (0.2 mM for AsPC-1, 0.1 mM for SW1990) and CD133 + cells were sorted by flow cytometry. Expression of AMPKα, Akt, Erk1/2, and mTOR and phosphorylation of CD133 + cells were evaluated by western blotting and the results were quantified using ImageJ V.1.46r (National Institutes of Health). Significant decreases of phospho-ERK1/2 and phospho-mTOR expression and a significant increase of phospho-Akt expression were observed in the metformin treated cells. Error bars represent the standard deviation. * P

Techniques Used: Flow Cytometry, Cytometry, Expressing, Western Blot, Standard Deviation

44) Product Images from "Resveratrol alleviates alcoholic fatty liver in mice"

Article Title: Resveratrol alleviates alcoholic fatty liver in mice

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.90358.2008

Resveratrol stimulated hepatic AMPK activity in ethanol-fed mice. A : Western blots were performed using anti-phosphorylated-AMPKα (anti-p-AMPKα), anti-AMPKα, anti-p-AMPKβ, anti-AMPKβ, and anti-phosphorylated acetyl CoA carboxylase (P-ACC) antibodies from liver extracts of mice fed a low-fat diet, 200 mg·kg −1 ·day −1 resveratrol, or 400 mg·kg −1 ·day −1 resveratrol with or without ethanol. β-Actin protein band was used to confirm equal loading and to normalize the data. B : relative levels of p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, and P-ACC. Western blots were quantified by a PhosphorImager and MultiAnalyst (Bio-Rad) software analysis. All data are expressed as means ± SD; n = 4–8 animals. Means without a common letter differ, P
Figure Legend Snippet: Resveratrol stimulated hepatic AMPK activity in ethanol-fed mice. A : Western blots were performed using anti-phosphorylated-AMPKα (anti-p-AMPKα), anti-AMPKα, anti-p-AMPKβ, anti-AMPKβ, and anti-phosphorylated acetyl CoA carboxylase (P-ACC) antibodies from liver extracts of mice fed a low-fat diet, 200 mg·kg −1 ·day −1 resveratrol, or 400 mg·kg −1 ·day −1 resveratrol with or without ethanol. β-Actin protein band was used to confirm equal loading and to normalize the data. B : relative levels of p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, and P-ACC. Western blots were quantified by a PhosphorImager and MultiAnalyst (Bio-Rad) software analysis. All data are expressed as means ± SD; n = 4–8 animals. Means without a common letter differ, P

Techniques Used: Activity Assay, Mouse Assay, Western Blot, Software

45) Product Images from "Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation"

Article Title: Loss of the E3 ubiquitin ligase MKRN1 represses diet-induced metabolic syndrome through AMPK activation

Journal: Nature Communications

doi: 10.1038/s41467-018-05721-4

MKRN1 depletion stimulates glucose metabolism by activating AMPK signalling. Analysis of wild-type (WT) or MKRN1 -knockout ( MK1 − / − ) littermate primary mouse embryonic fibroblasts (MEFs) and HepG2 cells transduced with two independent siRNAs targeting MKRN1 (siMK1 #6 and siMK1 #7) or a control siRNA for 48 h. a Glucose consumption in MK1 − / − MEFs (left) or MKRN1-depleted HepG2 cells (right) was measured based on the absorption of 2-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy- d -glucose (2-NBDG) by the cells. b , c Intracellular levels of glycolytic and citric acid cycle intermediates were determined by capillary electrophoresis–time-of-flight mass spectrometry of the MEFs. Each bar represents the relative amount of a metabolite for WT MEFs. G6P glucose-6-phosphate, F6P fructose-6-phosphate, F1,6-BP fructose-1,6-bisphosphate, G3P glucose-3-phosphate, 1,3-BG 1,3-bisphosphoglycerate, 3PG 3-phosphoglycerate, 2PG 2-phosphoglycerate, AcCoA acetyl-CoA, α-KG α-ketoglutarate. d , f To validate the AMPK signalling pathway or mRNA levels of AMPKα subunits, immunoblotting or quantitative real-time PCR analysis was performed using MEFs ( d ) and HepG2 cells ( f ). Cell lysates were immunoblotted with antibodies against pAMPKα, AMPKα, pACC, ACC, MKRN1 and actin. e The mRNA levels of glycolytic or lipogenic enzymes in MEFs were analysed by quantitative real-time PCR. g , h FFA-induced steatosis in HepG2 cells. Oil Red O staining ( g ) and TG levels (scale bar = 100 µm (top), 50 µm (bottom) ( h ) of the cells treated with FFA/FFA-free bovine serum albumin (BSA), which served as controls. All the experiments with MEFs were conducted in cells within the first 3–6 passages. i Fatty acid oxidation was analysed in MKRN1-depleted HepG2 cells. j The basal oxygen consumption rate (OCR) was measured in WT and MK1 −/− MEFs. k After sequential treatment with oligomycin, FCCP and rotenone/antimycin-A in the presence of BSA or BSA conjugated to palmitate, OCR was measured in WT or MK1 − /− MEFs. The results were normalised against total protein levels using XF-Analyze. All data are presented as the mean ± standard deviation (s.d.) of triplicate samples and are representative of at least three independent experiments. two-tailed Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
Figure Legend Snippet: MKRN1 depletion stimulates glucose metabolism by activating AMPK signalling. Analysis of wild-type (WT) or MKRN1 -knockout ( MK1 − / − ) littermate primary mouse embryonic fibroblasts (MEFs) and HepG2 cells transduced with two independent siRNAs targeting MKRN1 (siMK1 #6 and siMK1 #7) or a control siRNA for 48 h. a Glucose consumption in MK1 − / − MEFs (left) or MKRN1-depleted HepG2 cells (right) was measured based on the absorption of 2-[ N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy- d -glucose (2-NBDG) by the cells. b , c Intracellular levels of glycolytic and citric acid cycle intermediates were determined by capillary electrophoresis–time-of-flight mass spectrometry of the MEFs. Each bar represents the relative amount of a metabolite for WT MEFs. G6P glucose-6-phosphate, F6P fructose-6-phosphate, F1,6-BP fructose-1,6-bisphosphate, G3P glucose-3-phosphate, 1,3-BG 1,3-bisphosphoglycerate, 3PG 3-phosphoglycerate, 2PG 2-phosphoglycerate, AcCoA acetyl-CoA, α-KG α-ketoglutarate. d , f To validate the AMPK signalling pathway or mRNA levels of AMPKα subunits, immunoblotting or quantitative real-time PCR analysis was performed using MEFs ( d ) and HepG2 cells ( f ). Cell lysates were immunoblotted with antibodies against pAMPKα, AMPKα, pACC, ACC, MKRN1 and actin. e The mRNA levels of glycolytic or lipogenic enzymes in MEFs were analysed by quantitative real-time PCR. g , h FFA-induced steatosis in HepG2 cells. Oil Red O staining ( g ) and TG levels (scale bar = 100 µm (top), 50 µm (bottom) ( h ) of the cells treated with FFA/FFA-free bovine serum albumin (BSA), which served as controls. All the experiments with MEFs were conducted in cells within the first 3–6 passages. i Fatty acid oxidation was analysed in MKRN1-depleted HepG2 cells. j The basal oxygen consumption rate (OCR) was measured in WT and MK1 −/− MEFs. k After sequential treatment with oligomycin, FCCP and rotenone/antimycin-A in the presence of BSA or BSA conjugated to palmitate, OCR was measured in WT or MK1 − /− MEFs. The results were normalised against total protein levels using XF-Analyze. All data are presented as the mean ± standard deviation (s.d.) of triplicate samples and are representative of at least three independent experiments. two-tailed Student’s t -test; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

Techniques Used: Knock-Out, Transduction, Electrophoresis, Mass Spectrometry, Real-time Polymerase Chain Reaction, Staining, Standard Deviation, Two Tailed Test

The E3 ubiquitin ligase MKRN1 ubiquitinates and degrades AMPKα subunits. a MKRN1 knockdown increases AMPKα1 protein levels. The HepG2 cells were transfected with MKRN1 siRNAs (#6 and #7). b MKRN1 knockout stabilises AMPKα. WT or MK1 −/− MEFs were treated with CHX (100 mg ml −1 ) at the indicated time points. c , d MKRN1 expression promotes the proteasomal degradation of AMPKα subunits. The protein levels of ectopically expressed AMPK subunits were analysed using HEK293T cells. GFP was used as a transfection control ( c ). HepG2 cells were infected with retrovirus expressing MKRN1, followed by selection using puromycin. The cells were treated with 20 µM of MG132 for 6 h, and AMPKα1, α2, MKRN1 and actin were detected with the indicated antibodies ( d ). e MKRN1 induces both AMPKα1 and α2 ubiquitination. Constructs expressing FLAG/AMPKα1, α2, β1, γ1, 3.1/MKRN1 and HA/Ub were transfected into 293T cells. The ubiquitination assay was performed using cell lysates under denaturing conditions (in 1% SDS buffer). f , g MKRN1 directly ubiquitinates AMPKα subunits. In vitro ubiquitination of AMPKα1 ( f ) and α2 ( g ). h , i MKRN1 is required for the ubiquitination of AMPKα. Ubiquitinated endogenous AMPKα was determined under denaturing conditions using MG132-treated MEFs ( h ) and HepG2 cells ( i ). All the experiments with MEFs were conducted in cells within the first 3–6 passages. The data are representative of at least three independent experiments
Figure Legend Snippet: The E3 ubiquitin ligase MKRN1 ubiquitinates and degrades AMPKα subunits. a MKRN1 knockdown increases AMPKα1 protein levels. The HepG2 cells were transfected with MKRN1 siRNAs (#6 and #7). b MKRN1 knockout stabilises AMPKα. WT or MK1 −/− MEFs were treated with CHX (100 mg ml −1 ) at the indicated time points. c , d MKRN1 expression promotes the proteasomal degradation of AMPKα subunits. The protein levels of ectopically expressed AMPK subunits were analysed using HEK293T cells. GFP was used as a transfection control ( c ). HepG2 cells were infected with retrovirus expressing MKRN1, followed by selection using puromycin. The cells were treated with 20 µM of MG132 for 6 h, and AMPKα1, α2, MKRN1 and actin were detected with the indicated antibodies ( d ). e MKRN1 induces both AMPKα1 and α2 ubiquitination. Constructs expressing FLAG/AMPKα1, α2, β1, γ1, 3.1/MKRN1 and HA/Ub were transfected into 293T cells. The ubiquitination assay was performed using cell lysates under denaturing conditions (in 1% SDS buffer). f , g MKRN1 directly ubiquitinates AMPKα subunits. In vitro ubiquitination of AMPKα1 ( f ) and α2 ( g ). h , i MKRN1 is required for the ubiquitination of AMPKα. Ubiquitinated endogenous AMPKα was determined under denaturing conditions using MG132-treated MEFs ( h ) and HepG2 cells ( i ). All the experiments with MEFs were conducted in cells within the first 3–6 passages. The data are representative of at least three independent experiments

Techniques Used: Transfection, Knock-Out, Expressing, Infection, Selection, Construct, Ubiquitin Assay, In Vitro

46) Product Images from "Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1"

Article Title: Metformin promotes antitumor immunity via endoplasmic reticulum-associated degradation of PD-L1

Journal: Molecular cell

doi: 10.1016/j.molcel.2018.07.030

Metformin increases CTL activity through the AMPK/PD-L1 axis. (A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm 2 in (D) and 262,144 μm 2 in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. * P , 0.01~0.05, ** P , 0.001~0.01, # P ,
Figure Legend Snippet: Metformin increases CTL activity through the AMPK/PD-L1 axis. (A) 4T1_luc2 cells were injected into mice (n = 6 mice per group) on day 0, and metformin administered as indicated. (B) Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) Quantification (top) of bioluminescence imaging. Bottom, endpoint images shown (D) Immunostaining of cleaved caspase 3 in the 4T1 tumor mass. Hoechst: nuclear counter staining (pseudo-color: green). Scale bar, 200 μm (inset, 50 μm). (E) Immunostaining of CD8 (CTL marker) and granzyme B (activity of T cell) in the 4T1 tumor mass. Scale bar, 200 μm (inset, 50 μm). For (D) and (E), data represent mean ± SD. n = 12; 3 tissue slides per tumor, 4 mice per group. Unit = 466,038 μm 2 in (D) and 262,144 μm 2 in (E). (F) T cell-mediated cancer cell killing assay. MDA-MB-231 WT and AMPKα KO cells co-cultured with activated T cell for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T-cell ratio, 1:5. Data represent mean ± SD. n = 3. (G) Correlation analysis between PD-L1 and AMPKα T172-p expression in 14 breast cancer cell lines. (H) BT-549 and MDA-MB-231 were treated with increasing concentrations of metformin (1.25 to 5 mM) for 24 hr. (I) MDA-MB-231 parental, AMPKα WT and KO cells were treated with metformin (5 mM) for 24 hr. Compound C (Com C) AMPK inhibitor was pretreated 6 hr before metformin treatment. (J) Membrane PD-L1 expression by flow cytometric analysis after metformin (5 mM) treatment for 24 hr. (K) Right, quantitation of binding of green fluorescent-labeled PD-1/Fc on MDA-MB-231 treated with or without metformin for 24 hr. Data represent mean ± SD. n = 3. Left, representative images shown. Scale bar, 100 μm. (L) MDA-MB-231 WT and PD-L1 KO cells co-cultured with activated T cells for 48 hr with or without metformin (5 mM) were subjected to crystal violet staining. MDA-MB-231 to T- cell ratio, 1:3. * P , 0.01~0.05, ** P , 0.001~0.01, # P ,

Techniques Used: CTL Assay, Activity Assay, Injection, Mouse Assay, Imaging, Immunostaining, Staining, Marker, Multiple Displacement Amplification, Cell Culture, Expressing, Flow Cytometry, Quantitation Assay, Binding Assay, Labeling

The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant (A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm 2 . (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. * P , 0.01~0.05, ** P , 0.001~0.01, and # P ,
Figure Legend Snippet: The reduction of PD-L1 by metformin-activated AMPK is physiologically significant and clinically relevant (A) 4T1 WT or S194E mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) (B) 4T1 WT or S194A mPD-L1 stable cells (5 × 10 4 ) were injected into BALB/c mice (n = 7 mice per group) on day 0, and metformin (200 mg/kg) administered by i.p. injection starting at day 4 for 18 days. Tumor volume was measured on the indicated time points. Data represent mean ± SD. (C) The relative tumor volume (%) at the end point (day 22) of panel (B). (D) Left, representative images of immunostaining of CD8 and GB in the 4T1 tumor mass. Hoechst: counter staining. Right, CD8 and GB were quantified using Image J. Data represent mean ± SD. n = 12. Three tissue slides per tumor, 4 mice per group. Unit = 262,144 μm 2 . (E) AMPKα T172-p and PD-L1 levels before and after metformintreatment in tumor tissues of breast cancer patients. −, score 0; +, score 1; ++, score 2; +++, score 3; ↑, up; ↓, down; X, no change. (F) Representative IHC images of the cases shown in panel (E). Scale bar, 50 μm. (G) Plot of IHC scores of AMPKα T172-p and PD-L1 expression levels in responders before and after metformin treatment. n = 7 (H) PBS and metformin were administered to a pair of SCID mice with same patient tumor for 7 days. Immunostaining of AMPKα T172-p and PD-L1 in the PDX tumor mass. Hoechst:counter staining. Scale bar, 200 μm. (I) Western blotting of lysates from PDX tumors in (H) with the indicated antibodies. * P , 0.01~0.05, ** P , 0.001~0.01, and # P ,

Techniques Used: Injection, Mouse Assay, Immunostaining, Staining, Immunohistochemistry, Expressing, Western Blot

Related Articles

Clone Assay:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: .. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells. ..

Centrifugation:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Frozen tissues were homogenized with a polytron homogenizer on ice in RIPA buffer [20 m m tris–HCl, pH 7.5, 150 m m NaCl, 1 m m ethylenediamine tetraacetic acid, 1.0% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecylsulfate] supplemented with PhosSTOP phosphatase inhibitor cocktail and Complete protease inhibitor cocktail (Roche, Indianapolis, IN), followed by centrifugation at 13,200 g for 30 min. .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Article Title: Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens
Article Snippet: Western Blotting Skeletal muscles of 90-day-old chickens were cut into small pieces and resuspended in RIPA lysis and extraction buffer (Thermo Fisher Scientific) for 30 min at 4 °C prior to centrifugation for 10 min at 12,000× g . .. The following antibodies were used: anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA; 1:1000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-ATP5A (rabbit polyclonal; Abcam, Cambridge, UK; 1:250), anti-GHR (rabbit polyclonal; Bioss, Woburn, MA, USA; 1:500), anti-IGFBP2 (rabbit polyclonal; Bioss; 1:500), anti-PRX III (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), anti-beta actin (rabbit polyclonal; Bioss; 1:1000), goat anti-mouse (Santa Cruz Biotechnology; 1: 5000), and goat anti-rabbit (Abcam; 1: 5000) IgG coupled to horseradish peroxidase conjugate.

Stable Transfection:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: Paragraph title: Generation of stable cell Lines (AMPKα1S173C, AMPKα1S173D and AMPKα1WT) ... Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

Pyrolysis Gas Chromatography:

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: .. Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China). .. After incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.), the bands were detected by ECL chemiluminescence (Molecular Imager ChemiDoc XRS+ System Bio-Rad, Hercules, CA, USA).

Blocking Assay:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA). .. FLCN mouse monoclonal antibody was raised against recombinant FLCN protein ( ). α-tubulin antibody was purchased from Sigma-Aldrich (St Louis MO).

Article Title: Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation
Article Snippet: .. The membranes were blocking with non-fat dry milk for 1 h and incubated at room temperature for 2 h with p-LKB1, LKB1, p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, p-ACC, ACC (Cell Signaling Technology, Boston, MA, USA) or SREBP-1, CYP2E1, GAPDH, Sirt1, PPARγ (Abcam, Cambridge, MA, USA) or caspase-1, β-actin, PPARα (Santa Cruz, CA, USA) or IL-1β (R & D Systems Europe Ltd., Abingdon, UK). .. Then the membranes were followed by incubated with HRP-conjugated secondary antibody for 1 h at room temperature and visualized by ECL Prime Western Blotting Detection Reagent (Bio-Rad, USA).

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated with primary polyclonal IgG for nNOS (1:1000, Abcam. .. Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China).

Enzyme-linked Immunosorbent Assay:

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation
Article Snippet: The ELISA Ready-SET-Go Kits for TNF-α and IL-10 were purchased from eBiosciences (San Diego, CA, United States). .. AMPKα, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), p-CaMKKβ (Ser511), ACC, p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); iNOS, CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); CD68, CD206, and YM1/2 were purchased from Abcam (Cambridge, MA, United States).

Incubation:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA). .. FLCN mouse monoclonal antibody was raised against recombinant FLCN protein ( ). α-tubulin antibody was purchased from Sigma-Aldrich (St Louis MO).

Article Title: Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation
Article Snippet: .. The membranes were blocking with non-fat dry milk for 1 h and incubated at room temperature for 2 h with p-LKB1, LKB1, p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, p-ACC, ACC (Cell Signaling Technology, Boston, MA, USA) or SREBP-1, CYP2E1, GAPDH, Sirt1, PPARγ (Abcam, Cambridge, MA, USA) or caspase-1, β-actin, PPARα (Santa Cruz, CA, USA) or IL-1β (R & D Systems Europe Ltd., Abingdon, UK). .. Then the membranes were followed by incubated with HRP-conjugated secondary antibody for 1 h at room temperature and visualized by ECL Prime Western Blotting Detection Reagent (Bio-Rad, USA).

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: After blocking with 5% bovine serum albumin, membranes were incubated with primary polyclonal IgG for nNOS (1:1000, Abcam. .. Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China).

Activity Assay:

Article Title: Targeted Expression of Catalase to Mitochondria Prevents Age-Associated Reductions in Mitochondrial Function and Insulin Resistance
Article Snippet: AKT2 activity and PKCθ membrane translocation were assessed in protein extracts from gastrocnemius and quadriceps muscle, respectively, harvested after hyperinsulinemic-euglycemic clamp study using the methods previously described ( ; ; ). .. The primary antibodies used in the current study were as follows: PKCθ (Santa Cruz), p-AMPKT172 (Cell Signaling), AMPKα (Cell Signaling), pan-actin (Cell signaling), GAPDH (Cell signaling), PGC1α (Santacruz), phospho-ACC2S212 (Santacruz) and Catalase (Abcam).

Expressing:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: .. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells. ..

Article Title: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
Article Snippet: .. We checked the expression level of AKIN10 protein by Western blotting using two antibodies: firstly the AKIN10 specific antibody from Agrisera (AS 10919), and secondly an antibody against the phosphorylated T‐loop threonine (T172) of AMPKα, the mammalian AKIN10 ortholog (Cell Signaling #2531), that specifically recognizes the phosphorylated T‐loop threonine in AKIN10 and AKIN11 (T175 and T176, respectively; Baena‐González et al., 2007). .. Notably both antibodies bind to regions far before the T‐DNA insertion in the AKIN10 sequence.

BIA-KA:

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation
Article Snippet: RPMI 1640, DMEM, OPTI medium, FBS, penicillin, streptomycin, RNA iMAX Kit, DAPI solution, and BCA kit were purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). .. AMPKα, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), p-CaMKKβ (Ser511), ACC, p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); iNOS, CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); CD68, CD206, and YM1/2 were purchased from Abcam (Cambridge, MA, United States).

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Protein concentrations of cleared supernatants were measured with BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and adjusted to 1.33 mg/ml. .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells
Article Snippet: .. After the whole-cell protein extracts were quantified by a bicinchoninic acid (BCA) assay, the extracts were separated on NuPAGE 10%–12% polyacrylamide gels, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), blocked in 5% BSA in TBST, and hybridized with antibodies against β-actin (Abcam, 1:4 000), TNSALP (Abcam; R & D Systems, 1:500), p16INK4α (Abcam, 1:1 000), p53 (Cell Signaling, 1:1 000), Runx2 (Cell Signaling, 1:1 000), OCN (Santa Cruz, 1:800), PPAR-γ (Abcam, 1:300), AMPKα (Cell Signaling, 1:1 000), phospho-AMPKα (Thr172) (Cell Signaling, 1:1 000), ACC (Cell Signaling, 1:1 000), phospho-ACC (Cell Signaling, 1:1 000), CD73 (Cell Signaling, 1:1 000) and CD39 (Proteintech, 1:800). β-Actin was used as a loading control. ..

Modification:

Article Title: Cardioprotection by combination of three compounds from ShengMai preparations in mice with myocardial ischemia/reperfusion injury through AMPK activation-mediated mitochondrial fission
Article Snippet: Dulbecco’s modified Eagle medium (DMEM) was obtained from GIBCO/BRL (Life Technologies, California, USA). .. Antibody against β-actin was from Bioworld Technology (St. Louis Park, MN, USA), antibody against caspase-3, Bcl-2, Bax, Drp1, phospho-Drp1 (Ser616), AMPKα and phospho-AMPKα (Thr-172) were obtained from Cell Signaling Technology (Boston, MA, USA).

Western Blot:

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation
Article Snippet: AMPKα, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), p-CaMKKβ (Ser511), ACC, p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); iNOS, CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); CD68, CD206, and YM1/2 were purchased from Abcam (Cambridge, MA, United States). .. ECLplus Western Blotting Detection Reagents Kit was purchased from GE Healthcare (NJ, United States).

Article Title: Targeted Expression of Catalase to Mitochondria Prevents Age-Associated Reductions in Mitochondrial Function and Insulin Resistance
Article Snippet: Immunoblots were quantified from multiple exposures using ImageJ (NIH). .. The primary antibodies used in the current study were as follows: PKCθ (Santa Cruz), p-AMPKT172 (Cell Signaling), AMPKα (Cell Signaling), pan-actin (Cell signaling), GAPDH (Cell signaling), PGC1α (Santacruz), phospho-ACC2S212 (Santacruz) and Catalase (Abcam).

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Paragraph title: Protein isolation and western blotting ... In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Article Title: Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation
Article Snippet: Paragraph title: Western Blotting Analysis ... The membranes were blocking with non-fat dry milk for 1 h and incubated at room temperature for 2 h with p-LKB1, LKB1, p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, p-ACC, ACC (Cell Signaling Technology, Boston, MA, USA) or SREBP-1, CYP2E1, GAPDH, Sirt1, PPARγ (Abcam, Cambridge, MA, USA) or caspase-1, β-actin, PPARα (Santa Cruz, CA, USA) or IL-1β (R & D Systems Europe Ltd., Abingdon, UK).

Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells
Article Snippet: Paragraph title: Western blotting analysis ... After the whole-cell protein extracts were quantified by a bicinchoninic acid (BCA) assay, the extracts were separated on NuPAGE 10%–12% polyacrylamide gels, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), blocked in 5% BSA in TBST, and hybridized with antibodies against β-actin (Abcam, 1:4 000), TNSALP (Abcam; R & D Systems, 1:500), p16INK4α (Abcam, 1:1 000), p53 (Cell Signaling, 1:1 000), Runx2 (Cell Signaling, 1:1 000), OCN (Santa Cruz, 1:800), PPAR-γ (Abcam, 1:300), AMPKα (Cell Signaling, 1:1 000), phospho-AMPKα (Thr172) (Cell Signaling, 1:1 000), ACC (Cell Signaling, 1:1 000), phospho-ACC (Cell Signaling, 1:1 000), CD73 (Cell Signaling, 1:1 000) and CD39 (Proteintech, 1:800). β-Actin was used as a loading control.

Article Title: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
Article Snippet: .. We checked the expression level of AKIN10 protein by Western blotting using two antibodies: firstly the AKIN10 specific antibody from Agrisera (AS 10919), and secondly an antibody against the phosphorylated T‐loop threonine (T172) of AMPKα, the mammalian AKIN10 ortholog (Cell Signaling #2531), that specifically recognizes the phosphorylated T‐loop threonine in AKIN10 and AKIN11 (T175 and T176, respectively; Baena‐González et al., 2007). .. Notably both antibodies bind to regions far before the T‐DNA insertion in the AKIN10 sequence.

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: Paragraph title: 3.8. Western Blot ... Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China).

Article Title: Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens
Article Snippet: Paragraph title: 4.6. Western Blotting ... The following antibodies were used: anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA; 1:1000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-ATP5A (rabbit polyclonal; Abcam, Cambridge, UK; 1:250), anti-GHR (rabbit polyclonal; Bioss, Woburn, MA, USA; 1:500), anti-IGFBP2 (rabbit polyclonal; Bioss; 1:500), anti-PRX III (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), anti-beta actin (rabbit polyclonal; Bioss; 1:1000), goat anti-mouse (Santa Cruz Biotechnology; 1: 5000), and goat anti-rabbit (Abcam; 1: 5000) IgG coupled to horseradish peroxidase conjugate.

Article Title: Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles
Article Snippet: Paragraph title: Western Blotting ... Values of immunoblotting bands were normalized using total protein values. p-ERK (Thr202/Tyr204), ERK, p-AMPKα (Thr172), AMPKα, p-mTOR (Ser2481), mTOR, p-AKT (Ser473), AKT, p-AS160 (Thr642), AS160, and p-PKCδ/θ (Ser643/676) antibodies were purchased from Cell Signaling.

Translocation Assay:

Article Title: Targeted Expression of Catalase to Mitochondria Prevents Age-Associated Reductions in Mitochondrial Function and Insulin Resistance
Article Snippet: AKT2 activity and PKCθ membrane translocation were assessed in protein extracts from gastrocnemius and quadriceps muscle, respectively, harvested after hyperinsulinemic-euglycemic clamp study using the methods previously described ( ; ; ). .. The primary antibodies used in the current study were as follows: PKCθ (Santa Cruz), p-AMPKT172 (Cell Signaling), AMPKα (Cell Signaling), pan-actin (Cell signaling), GAPDH (Cell signaling), PGC1α (Santacruz), phospho-ACC2S212 (Santacruz) and Catalase (Abcam).

Colorimetric Assay:

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation
Article Snippet: NO Colorimetric Assay Kit was purchased from BioVision (Milpitas, CA, United States). .. AMPKα, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), p-CaMKKβ (Ser511), ACC, p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); iNOS, CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); CD68, CD206, and YM1/2 were purchased from Abcam (Cambridge, MA, United States).

Transfection:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: After 24 h, the antibiotic Geneticin (500 μg/ml) (Invitrogen, Thermo Fisher Scientific) was added to the media in order to select the transfected cells. .. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

Protease Inhibitor:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Frozen tissues were homogenized with a polytron homogenizer on ice in RIPA buffer [20 m m tris–HCl, pH 7.5, 150 m m NaCl, 1 m m ethylenediamine tetraacetic acid, 1.0% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecylsulfate] supplemented with PhosSTOP phosphatase inhibitor cocktail and Complete protease inhibitor cocktail (Roche, Indianapolis, IN), followed by centrifugation at 13,200 g for 30 min. .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Article Title: Cardioprotection by combination of three compounds from ShengMai preparations in mice with myocardial ischemia/reperfusion injury through AMPK activation-mediated mitochondrial fission
Article Snippet: RIPA lysis buffer, protease inhibitor and enhanced chemiluminescene (ECL) reagent were from Vazyme Biotech (Nanjing, China). .. Antibody against β-actin was from Bioworld Technology (St. Louis Park, MN, USA), antibody against caspase-3, Bcl-2, Bax, Drp1, phospho-Drp1 (Ser616), AMPKα and phospho-AMPKα (Thr-172) were obtained from Cell Signaling Technology (Boston, MA, USA).

Imaging:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Immunoblotting was performed as previously described ( ) with minor modifications optimized for the Odyssey infrared imaging system (LI-COR, Lincoln, NE). .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Article Title: Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles
Article Snippet: Bands were identified using ChemiDoc Imaging System, using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo, MA, USA). .. Values of immunoblotting bands were normalized using total protein values. p-ERK (Thr202/Tyr204), ERK, p-AMPKα (Thr172), AMPKα, p-mTOR (Ser2481), mTOR, p-AKT (Ser473), AKT, p-AS160 (Thr642), AS160, and p-PKCδ/θ (Ser643/676) antibodies were purchased from Cell Signaling.

Protein Concentration:

Article Title: Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens
Article Snippet: The protein concentration was measured using a bicinchoninic acid protein assay kit (Sigma-Aldrich) and adjusted to equal protein concentration. .. The following antibodies were used: anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA; 1:1000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-ATP5A (rabbit polyclonal; Abcam, Cambridge, UK; 1:250), anti-GHR (rabbit polyclonal; Bioss, Woburn, MA, USA; 1:500), anti-IGFBP2 (rabbit polyclonal; Bioss; 1:500), anti-PRX III (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), anti-beta actin (rabbit polyclonal; Bioss; 1:1000), goat anti-mouse (Santa Cruz Biotechnology; 1: 5000), and goat anti-rabbit (Abcam; 1: 5000) IgG coupled to horseradish peroxidase conjugate.

Polymerase Chain Reaction:

Article Title: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
Article Snippet: This was tested by PCR using gene‐specific primers against the flanking region of the second insertion but no insertion could be detected ( ). .. We checked the expression level of AKIN10 protein by Western blotting using two antibodies: firstly the AKIN10 specific antibody from Agrisera (AS 10919), and secondly an antibody against the phosphorylated T‐loop threonine (T172) of AMPKα, the mammalian AKIN10 ortholog (Cell Signaling #2531), that specifically recognizes the phosphorylated T‐loop threonine in AKIN10 and AKIN11 (T175 and T176, respectively; Baena‐González et al., 2007).

Recombinant:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA). .. FLCN mouse monoclonal antibody was raised against recombinant FLCN protein ( ). α-tubulin antibody was purchased from Sigma-Aldrich (St Louis MO).

Nucleic Acid Electrophoresis:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Four times sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS) sample buffer was then added and samples were boiled for 5 min to produce 1 μg/μl sample lysates. .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Article Title: Acanthoic Acid Can Partially Prevent Alcohol Exposure-Induced Liver Lipid Deposition and Inflammation
Article Snippet: Briefly, protein samples were placed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. .. The membranes were blocking with non-fat dry milk for 1 h and incubated at room temperature for 2 h with p-LKB1, LKB1, p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, p-ACC, ACC (Cell Signaling Technology, Boston, MA, USA) or SREBP-1, CYP2E1, GAPDH, Sirt1, PPARγ (Abcam, Cambridge, MA, USA) or caspase-1, β-actin, PPARα (Santa Cruz, CA, USA) or IL-1β (R & D Systems Europe Ltd., Abingdon, UK).

Electroporation:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: C3A cells were transfected by electroporation, as previously described [ ]. .. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

Mutagenesis:

Article Title: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
Article Snippet: This mutant has been used – and confirmed – by three groups in our consortium independently. shows clearly that this mutant (GABI KAT GABI_579E09 ) is indeed a valid akin10 null mutant. .. We checked the expression level of AKIN10 protein by Western blotting using two antibodies: firstly the AKIN10 specific antibody from Agrisera (AS 10919), and secondly an antibody against the phosphorylated T‐loop threonine (T172) of AMPKα, the mammalian AKIN10 ortholog (Cell Signaling #2531), that specifically recognizes the phosphorylated T‐loop threonine in AKIN10 and AKIN11 (T175 and T176, respectively; Baena‐González et al., 2007).

Isolation:

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation
Article Snippet: High Pure RNA Isolation Kit, Transcriptor First Strand cDNA Synthesis Kit, and FastStart Universal SYBR Green Master Reagents were purchased from Roche Applied Science (Mannheim, Germany). .. AMPKα, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), p-CaMKKβ (Ser511), ACC, p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); iNOS, CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); CD68, CD206, and YM1/2 were purchased from Abcam (Cambridge, MA, United States).

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: Paragraph title: Protein isolation and western blotting ... In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Bicinchoninic Acid Protein Assay:

Article Title: Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens
Article Snippet: The protein concentration was measured using a bicinchoninic acid protein assay kit (Sigma-Aldrich) and adjusted to equal protein concentration. .. The following antibodies were used: anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA; 1:1000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-ATP5A (rabbit polyclonal; Abcam, Cambridge, UK; 1:250), anti-GHR (rabbit polyclonal; Bioss, Woburn, MA, USA; 1:500), anti-IGFBP2 (rabbit polyclonal; Bioss; 1:500), anti-PRX III (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), anti-beta actin (rabbit polyclonal; Bioss; 1:1000), goat anti-mouse (Santa Cruz Biotechnology; 1: 5000), and goat anti-rabbit (Abcam; 1: 5000) IgG coupled to horseradish peroxidase conjugate.

Immunodetection:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: .. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells. ..

Sequencing:

Article Title: SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants
Article Snippet: We checked the expression level of AKIN10 protein by Western blotting using two antibodies: firstly the AKIN10 specific antibody from Agrisera (AS 10919), and secondly an antibody against the phosphorylated T‐loop threonine (T172) of AMPKα, the mammalian AKIN10 ortholog (Cell Signaling #2531), that specifically recognizes the phosphorylated T‐loop threonine in AKIN10 and AKIN11 (T175 and T176, respectively; Baena‐González et al., 2007). .. Notably both antibodies bind to regions far before the T‐DNA insertion in the AKIN10 sequence.

Polyacrylamide Gel Electrophoresis:

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (10%) and transferred to nitrocellulose membranes. .. Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China).

Lysis:

Article Title: Cardioprotection by combination of three compounds from ShengMai preparations in mice with myocardial ischemia/reperfusion injury through AMPK activation-mediated mitochondrial fission
Article Snippet: RIPA lysis buffer, protease inhibitor and enhanced chemiluminescene (ECL) reagent were from Vazyme Biotech (Nanjing, China). .. Antibody against β-actin was from Bioworld Technology (St. Louis Park, MN, USA), antibody against caspase-3, Bcl-2, Bax, Drp1, phospho-Drp1 (Ser616), AMPKα and phospho-AMPKα (Thr-172) were obtained from Cell Signaling Technology (Boston, MA, USA).

Article Title: Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells
Article Snippet: Western blotting analysis The MSCs were harvested in RIPA lysis buffer (Beyotime Co., Shanghai, China). .. After the whole-cell protein extracts were quantified by a bicinchoninic acid (BCA) assay, the extracts were separated on NuPAGE 10%–12% polyacrylamide gels, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), blocked in 5% BSA in TBST, and hybridized with antibodies against β-actin (Abcam, 1:4 000), TNSALP (Abcam; R & D Systems, 1:500), p16INK4α (Abcam, 1:1 000), p53 (Cell Signaling, 1:1 000), Runx2 (Cell Signaling, 1:1 000), OCN (Santa Cruz, 1:800), PPAR-γ (Abcam, 1:300), AMPKα (Cell Signaling, 1:1 000), phospho-AMPKα (Thr172) (Cell Signaling, 1:1 000), ACC (Cell Signaling, 1:1 000), phospho-ACC (Cell Signaling, 1:1 000), CD73 (Cell Signaling, 1:1 000) and CD39 (Proteintech, 1:800). β-Actin was used as a loading control.

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: Protein lysates were obtained by homogenizing sciatic nerves with lysis buffer. .. Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China).

Article Title: Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens
Article Snippet: Western Blotting Skeletal muscles of 90-day-old chickens were cut into small pieces and resuspended in RIPA lysis and extraction buffer (Thermo Fisher Scientific) for 30 min at 4 °C prior to centrifugation for 10 min at 12,000× g . .. The following antibodies were used: anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA; 1:1000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-ATP5A (rabbit polyclonal; Abcam, Cambridge, UK; 1:250), anti-GHR (rabbit polyclonal; Bioss, Woburn, MA, USA; 1:500), anti-IGFBP2 (rabbit polyclonal; Bioss; 1:500), anti-PRX III (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), anti-beta actin (rabbit polyclonal; Bioss; 1:1000), goat anti-mouse (Santa Cruz Biotechnology; 1: 5000), and goat anti-rabbit (Abcam; 1: 5000) IgG coupled to horseradish peroxidase conjugate.

SDS Page:

Article Title: Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation
Article Snippet: A total of 20 μg of protein was loaded onto 4–20% tris–glycine SDS-PAGE gels. .. In brief, separated proteins were transferred to Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked with Odyssey blocking buffer (LI-COR) or 5% milk at room temperature for 1 h. Blocked membranes were incubated overnight at 4°C with primary antibodies, diluted with 0.1% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST; 20 m m Tris–HCl pH8.0, 150 m m NaCl, 0.05% Tween 20) as follows: p-mTOR (S2448) 1:1000, total mTOR 1:500, p-Raptor(S792) 1:1000, p-AMPKα (T172) 1:1000, AMPKα 1:1000, p-S6 ribosomal protein (S240/244) 1:1000, total S6R 1:500, p-P70-S6 Kinase (T421/S424) 1:1000, p-4EBP1 (T37/46) 1:1000, total 4EBP1 1:500, glyceraldehyde-3-phosphate dehydrogenase 1:1000, p-AKT (T308) 1:1000, p-PDK1 (S241), p-ULK1 (S555) 1:1000, SQSTM1 1:1000, and LC3 (D11XP) 1;1000 (Cell Signaling, Danvers, MA).

Plasmid Preparation:

Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation
Article Snippet: Generation of stable cell Lines (AMPKα1S173C, AMPKα1S173D and AMPKα1WT) pCDNA3 plasmid harbouring Myc-AMPKα1(WT), Myc-AMPKα1(S173C) or Myc-AMPKα1(S173D) [ , ], kindly given by Dr. Dietbert Neumann (Maastricht University, The Netherlands), were used to generate populations of C3A cells which stably express mutated forms of the AMPKα1(S173) residue. .. Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

Software:

Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis
Article Snippet: Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China). .. The band size and density were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).

Article Title: Innovative Approach of Non-Thermal Plasma Application for Improving the Growth Rate in Chickens
Article Snippet: The following antibodies were used: anti-phospho-AMPKα (Thr172, p-AMPKα; rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA; 1:1000), anti-AMPKα (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-phospho-mTOR (Ser2448, p-mTOR; rabbit monoclonal; Cell Signaling Technology; 1:1000), anti-mTOR (rabbit polyclonal; Cell Signaling Technology; 1:1000), anti-ATP5A (rabbit polyclonal; Abcam, Cambridge, UK; 1:250), anti-GHR (rabbit polyclonal; Bioss, Woburn, MA, USA; 1:500), anti-IGFBP2 (rabbit polyclonal; Bioss; 1:500), anti-PRX III (mouse monoclonal; Santa Cruz Biotechnology, Dallas, TX, USA; 1:200), anti-beta actin (rabbit polyclonal; Bioss; 1:1000), goat anti-mouse (Santa Cruz Biotechnology; 1: 5000), and goat anti-rabbit (Abcam; 1: 5000) IgG coupled to horseradish peroxidase conjugate. .. Band intensity was quantified using the ImageJ software (National Institutes of Health, Bethesda, MA, USA).

Article Title: Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles
Article Snippet: Band densities of both stain-free blot and immunoblotting were measured using Image Lab Software (Bio-Rad). .. Values of immunoblotting bands were normalized using total protein values. p-ERK (Thr202/Tyr204), ERK, p-AMPKα (Thr172), AMPKα, p-mTOR (Ser2481), mTOR, p-AKT (Ser473), AKT, p-AS160 (Thr642), AS160, and p-PKCδ/θ (Ser643/676) antibodies were purchased from Cell Signaling.

SYBR Green Assay:

Article Title: Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation
Article Snippet: High Pure RNA Isolation Kit, Transcriptor First Strand cDNA Synthesis Kit, and FastStart Universal SYBR Green Master Reagents were purchased from Roche Applied Science (Mannheim, Germany). .. AMPKα, p-AMPK (Thr172), LKB1, p-LKB1 (Ser428), p-CaMKKβ (Ser511), ACC, p-ACC (Ser79), β-actin, GAPDH, and HRP-linked secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, United States); iNOS, CaMKKβ, and Arg-1 were purchased from Invitrogen (Carlsbad, CA, United States); CD68, CD206, and YM1/2 were purchased from Abcam (Cambridge, MA, United States).

Negative Control:

Article Title: Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma
Article Snippet: Antibodies and reagents Antibodies against the eukaryotic translation initiation factor 4E binding protein 1 (4EBP-1), P-4EBP-1 (Thr37/46), the ribosomal protein S6 (S6), P-S6 (Ser240/244), β-tubulin, AMPKα, P-AMPKα (Thr172), lactate dehydrogenase A (LDH-A), P-LDH-A (Tyr10), hexokinase II (HEXII), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (BAX) were purchased from Cell Signalling Technologies (Danvers, MA). .. BAX-inhibiting peptide V5 (Bax-V5), BAX-inhibiting negative control peptide (NCP), and Hoechst 33258 were purchased from MilliporeSigma (Burlington, MA).

Binding Assay:

Article Title: Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma
Article Snippet: .. Antibodies and reagents Antibodies against the eukaryotic translation initiation factor 4E binding protein 1 (4EBP-1), P-4EBP-1 (Thr37/46), the ribosomal protein S6 (S6), P-S6 (Ser240/244), β-tubulin, AMPKα, P-AMPKα (Thr172), lactate dehydrogenase A (LDH-A), P-LDH-A (Tyr10), hexokinase II (HEXII), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X (BAX) were purchased from Cell Signalling Technologies (Danvers, MA). .. Antibodies against BCATm and BCATc were previously described., Rapamycin and N -acetyl-leucine amide (NALA) were purchased from LC Laboratories (Woburn, MA) and BACHEM (Bubendorf, Switzerland), respectively.

Electrophoresis:

Article Title: Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles
Article Snippet: Equal amounts of protein were subjected to electrophoresis on Criterion stain-free precast gels (10%; Bio-Rad) and then transferred electrophoretically using Trans-Blot Turbo Transfer System (Bio-Rad) onto a polyvinylidene fluoride membrane (Bio-Rad). .. Values of immunoblotting bands were normalized using total protein values. p-ERK (Thr202/Tyr204), ERK, p-AMPKα (Thr172), AMPKα, p-mTOR (Ser2481), mTOR, p-AKT (Ser473), AKT, p-AS160 (Thr642), AS160, and p-PKCδ/θ (Ser643/676) antibodies were purchased from Cell Signaling.

Staining:

Article Title: Recombinant Uncarboxylated Osteocalcin Per Se Enhances Mouse Skeletal Muscle Glucose Uptake in both Extensor Digitorum Longus and Soleus Muscles
Article Snippet: Band densities of both stain-free blot and immunoblotting were measured using Image Lab Software (Bio-Rad). .. Values of immunoblotting bands were normalized using total protein values. p-ERK (Thr202/Tyr204), ERK, p-AMPKα (Thr172), AMPKα, p-mTOR (Ser2481), mTOR, p-AKT (Ser473), AKT, p-AS160 (Thr642), AS160, and p-PKCδ/θ (Ser643/676) antibodies were purchased from Cell Signaling.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cell Signaling Technology Inc ampkα
    AMPK activity and mitochondrial density in skeletal muscle in young and old WT and MCAT mice. (A) Representative immunoblots for each group and the ratio of p-AMPK T172 to <t>AMPKα,</t> the ratio of p-ACC2 Ser212 and PGC1α to actin in quadriceps
    Ampkα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ampkα/product/Cell Signaling Technology Inc
    Average 99 stars, based on 55 article reviews
    Price from $9.99 to $1999.99
    ampkα - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc protein kinase ampkα
    Expression of proteins involved in insulin signaling in skeletal muscle Levels represent fold change ± standard error of the mean compared to HCC. Western blot analysis demonstrated that alcohol consumption increased expression of p-IRS1, IRS2, AKT, <t>AMPKα,</t> PPARα, Fox01, and GLUT4.
    Protein Kinase Ampkα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein kinase ampkα/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein kinase ampkα - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    Image Search Results


    AMPK activity and mitochondrial density in skeletal muscle in young and old WT and MCAT mice. (A) Representative immunoblots for each group and the ratio of p-AMPK T172 to AMPKα, the ratio of p-ACC2 Ser212 and PGC1α to actin in quadriceps

    Journal: Cell metabolism

    Article Title: Targeted Expression of Catalase to Mitochondria Prevents Age-Associated Reductions in Mitochondrial Function and Insulin Resistance

    doi: 10.1016/j.cmet.2010.11.004

    Figure Lengend Snippet: AMPK activity and mitochondrial density in skeletal muscle in young and old WT and MCAT mice. (A) Representative immunoblots for each group and the ratio of p-AMPK T172 to AMPKα, the ratio of p-ACC2 Ser212 and PGC1α to actin in quadriceps

    Article Snippet: The primary antibodies used in the current study were as follows: PKCθ (Santa Cruz), p-AMPKT172 (Cell Signaling), AMPKα (Cell Signaling), pan-actin (Cell signaling), GAPDH (Cell signaling), PGC1α (Santacruz), phospho-ACC2S212 (Santacruz) and Catalase (Abcam).

    Techniques: Activity Assay, Mouse Assay, Western Blot

    Disruption of AMPKα1(S173) increases cell death during glucose starvation in hepatocarcinoma derived cells C3A cells stably expressing either AMPKα1(S173C) (S173C), AMPKα1(S173D) (S173D) or wild type AMPKα1(S173) (WT) were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. After 12 h of glucose deprivation cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172), AMPKα and Puma. α Tubulin was used as loading control. The blots in the picture are representative of 3 independent experiments. B. Cells attached to microplates were cultured for 0, 24, 48 and 72 h. MTT assay was performed as described in Materials and Methods . Results are presented as the percentage of the absorbance of the cells at 0 h. C. After 36 h cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent fold increase respect to WT controls (WT C) in the proportion of cells undergoing apoptosis (Annexin V positive). WT C apoptotic cells (mean value): 9%. Values represent the mean ± SEM of 3 experiments. * P

    Journal: Oncotarget

    Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    doi: 10.18632/oncotarget.7404

    Figure Lengend Snippet: Disruption of AMPKα1(S173) increases cell death during glucose starvation in hepatocarcinoma derived cells C3A cells stably expressing either AMPKα1(S173C) (S173C), AMPKα1(S173D) (S173D) or wild type AMPKα1(S173) (WT) were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. After 12 h of glucose deprivation cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172), AMPKα and Puma. α Tubulin was used as loading control. The blots in the picture are representative of 3 independent experiments. B. Cells attached to microplates were cultured for 0, 24, 48 and 72 h. MTT assay was performed as described in Materials and Methods . Results are presented as the percentage of the absorbance of the cells at 0 h. C. After 36 h cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent fold increase respect to WT controls (WT C) in the proportion of cells undergoing apoptosis (Annexin V positive). WT C apoptotic cells (mean value): 9%. Values represent the mean ± SEM of 3 experiments. * P

    Article Snippet: Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

    Techniques: Derivative Assay, Stable Transfection, Expressing, Incubation, Western Blot, Cell Culture, MTT Assay, Staining, Flow Cytometry, Cytometry

    AMPK and PKA activation during glucose restriction C3A cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). Cell lysates were obtained and analyzed by western blotting. A. Protein levels of P-AMPKα(T172) and AMPKα were detected at 2 and 6 h of glucose deprivation. B. Phosphorylated PKA substrates (RXXS/T-P residues) were detected after 2 h of treatments. Cells incubated with 100 μM dbcAMP (dbcAMP) were used as positive control of PKA phosphorylation. Bars represent band densities of PKA substrates weighted 37, 80 and 140 kDa. α Tubulin was used as loading control. Values represent the mean ± SEM of 3 experiments. * P

    Journal: Oncotarget

    Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    doi: 10.18632/oncotarget.7404

    Figure Lengend Snippet: AMPK and PKA activation during glucose restriction C3A cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). Cell lysates were obtained and analyzed by western blotting. A. Protein levels of P-AMPKα(T172) and AMPKα were detected at 2 and 6 h of glucose deprivation. B. Phosphorylated PKA substrates (RXXS/T-P residues) were detected after 2 h of treatments. Cells incubated with 100 μM dbcAMP (dbcAMP) were used as positive control of PKA phosphorylation. Bars represent band densities of PKA substrates weighted 37, 80 and 140 kDa. α Tubulin was used as loading control. Values represent the mean ± SEM of 3 experiments. * P

    Article Snippet: Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

    Techniques: Activation Assay, Incubation, Western Blot, Positive Control

    Effects of AMPK knock down on cell cycle progression and cell death during glucose deprivation in liver cancer cells C3A (A, B, C) HuH-7 and SK-Hep-1 (D) cells were transfected with a siRNA specifically targeted to AMPKα1 mRNA (AMPK KD), or with a siRNA control (Control), and allowed to grow for 48 hours. A. AMPKα expression was analyzed by western blot, in Control and AMPK KD cells. α Tubulin was used as loading control. In panels B, C and D control and AMPK KD cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0) during 24 h. B. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The figure shows typical outputs of the cytometric analysis and percentages of C3A cells in the different phases of the cell cycle, an experiment representative of 3 independent experiments. C. C3A cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent increase in the percentages of cells undergoing apoptosis (Annexin V positive) relative to controls (C). Control apoptotic cells (mean value): 8%. Values represent the mean ± SEM of 3 experiments. * P

    Journal: Oncotarget

    Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    doi: 10.18632/oncotarget.7404

    Figure Lengend Snippet: Effects of AMPK knock down on cell cycle progression and cell death during glucose deprivation in liver cancer cells C3A (A, B, C) HuH-7 and SK-Hep-1 (D) cells were transfected with a siRNA specifically targeted to AMPKα1 mRNA (AMPK KD), or with a siRNA control (Control), and allowed to grow for 48 hours. A. AMPKα expression was analyzed by western blot, in Control and AMPK KD cells. α Tubulin was used as loading control. In panels B, C and D control and AMPK KD cells were incubated with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0) during 24 h. B. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. The figure shows typical outputs of the cytometric analysis and percentages of C3A cells in the different phases of the cell cycle, an experiment representative of 3 independent experiments. C. C3A cells were stained with Annexin V-FITC and propidium iodide (PI) and the percentages of apoptotic cells were determined by flow cytometry analysis. Bar charts represent increase in the percentages of cells undergoing apoptosis (Annexin V positive) relative to controls (C). Control apoptotic cells (mean value): 8%. Values represent the mean ± SEM of 3 experiments. * P

    Article Snippet: Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

    Techniques: Transfection, Expressing, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry

    Regulation of AMPK activation by PKA phosphorylation in hepatic cancer cells C3A cells were incubated for 36 h (A) and 24 h (B) with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. C and Glc0 cells were incubated in the absence or presence of 100 μM dbcAMP (dbcAMP) or/and 1 mM AICAR (AICAR). Cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172) and AMPKα. β Actin was used as loading control. Bars represent the mean ± SEM of 3 experiments. * P

    Journal: Oncotarget

    Article Title: AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    doi: 10.18632/oncotarget.7404

    Figure Lengend Snippet: Regulation of AMPK activation by PKA phosphorylation in hepatic cancer cells C3A cells were incubated for 36 h (A) and 24 h (B) with 4.5 g/L glucose DMEM (C) or with no-glucose DMEM (Glc0). A. C and Glc0 cells were incubated in the absence or presence of 100 μM dbcAMP (dbcAMP) or/and 1 mM AICAR (AICAR). Cell lysates were obtained and analyzed by western blotting to determine protein levels of P-AMPKα(T172) and AMPKα. β Actin was used as loading control. Bars represent the mean ± SEM of 3 experiments. * P

    Article Snippet: Clones expressing the AMPKα1 mutants were identified by immunodetection of c-Myc tag (c-Myc Antibody (9E10), Santa Cruz Biotechnology Inc.) and AMPKα (Cell Signaling Technology) protein expression, and grown in a medium containing Geneticin 200 μg/ml in conditions otherwise similar to parental cells.

    Techniques: Activation Assay, Incubation, Western Blot

    Representative Western blot analyses of p-AMPKβ and AMPKβ ( A ), p-AMPKα and AMPKα ( B ) in rat sciatic nerves. Protein expression was analyzed by western blot, and bands were quantified by densitometry. β-Actin was used as a loading control and for relative quantification in all cases. # p

    Journal: Molecules

    Article Title: Salvianolic Acid A Protects the Peripheral Nerve Function in Diabetic Rats through Regulation of the AMPK-PGC1α-Sirt3 Axis

    doi: 10.3390/molecules170911216

    Figure Lengend Snippet: Representative Western blot analyses of p-AMPKβ and AMPKβ ( A ), p-AMPKα and AMPKα ( B ) in rat sciatic nerves. Protein expression was analyzed by western blot, and bands were quantified by densitometry. β-Actin was used as a loading control and for relative quantification in all cases. # p

    Article Snippet: Inc., Hong Kong, China) and AMPK signaling pathway-relevant proteins including AMPKα, phospho-AMPKα (p-AMPKα), AMPKβ, phospho-AMPKβ (p-AMPKβ), PGC-1α (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA), Sirt3 (1:400, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and LKB1 (1:1000, Beyotime, Inc., Beijing, China).

    Techniques: Western Blot, Expressing

    Expression of proteins involved in insulin signaling in skeletal muscle Levels represent fold change ± standard error of the mean compared to HCC. Western blot analysis demonstrated that alcohol consumption increased expression of p-IRS1, IRS2, AKT, AMPKα, PPARα, Fox01, and GLUT4.

    Journal: Surgery

    Article Title: Vodka And Wine Consumption In A Swine Model Of Metabolic Syndrome Alters Insulin Signaling Pathways In The Liver And Skeletal Muscle

    doi: 10.1016/j.surg.2012.06.014

    Figure Lengend Snippet: Expression of proteins involved in insulin signaling in skeletal muscle Levels represent fold change ± standard error of the mean compared to HCC. Western blot analysis demonstrated that alcohol consumption increased expression of p-IRS1, IRS2, AKT, AMPKα, PPARα, Fox01, and GLUT4.

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies at dilutions recommended by the manufacturer against phosphorylated insulin receptor substrate 1 (p-IRS1) (Ser612), IRS2, phosphorylated AKT (Thr 308), AMP- activated protein kinase (AMPKα), FoxO1 (all from Cell Signaling, Danvers, MA), peroxisome proliferating activated receptor α (PPARα) (Cayman Chemical, Ann Arbor, MI), and glucose transporter type 4 (GLUT4) (Epitomics, Burlingame, California).

    Techniques: Expressing, Western Blot

    Expression of proteins involved in hepatic insulin signaling Levels represent fold change ± standard error of the mean compared to HCC. Western blot analysis demonstrated that alcohol consumption moderately increased expression of AKT, AMPKα, and GLUT4.

    Journal: Surgery

    Article Title: Vodka And Wine Consumption In A Swine Model Of Metabolic Syndrome Alters Insulin Signaling Pathways In The Liver And Skeletal Muscle

    doi: 10.1016/j.surg.2012.06.014

    Figure Lengend Snippet: Expression of proteins involved in hepatic insulin signaling Levels represent fold change ± standard error of the mean compared to HCC. Western blot analysis demonstrated that alcohol consumption moderately increased expression of AKT, AMPKα, and GLUT4.

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies at dilutions recommended by the manufacturer against phosphorylated insulin receptor substrate 1 (p-IRS1) (Ser612), IRS2, phosphorylated AKT (Thr 308), AMP- activated protein kinase (AMPKα), FoxO1 (all from Cell Signaling, Danvers, MA), peroxisome proliferating activated receptor α (PPARα) (Cayman Chemical, Ann Arbor, MI), and glucose transporter type 4 (GLUT4) (Epitomics, Burlingame, California).

    Techniques: Expressing, Western Blot