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rabbit polyclonal antibody against pampk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antibody against pampk
    Rabbit Polyclonal Antibody Against Pampk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 5243 article reviews
    rabbit polyclonal antibody against pampk - by Bioz Stars, 2026-03
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    rabbit polyclonal antibody against pampk  (Cell Signaling Technology Inc)
    99
    Cell Signaling Technology Inc rabbit polyclonal antibody against pampk
    Rabbit Polyclonal Antibody Against Pampk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polyclonal anti rabbit pampkα  (Cell Signaling Technology Inc)
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    Luseogliflozin-mediated enhancement of master regulators of starvation and improvement in mitochondrial function, as shown by kidney metabolomic analysis at 1 week post ischemia/reperfusion (I/R) injury. ( a – d ) Western blot analysis and quantification of kidney expression of <t>pAMPK</t> ( a , b ) and Sirt3 ( c , d ) at 1 week post ischemia/reperfusion (I/R) injury in the following groups of mice: vehicle-treated + sham-operated (n = 6), luseogliflozin-treated + sham-operated (n = 6), vehicle-treated + I/R-injured (n = 6), and luseogliflozin-treated + I/R-injured (n=6). ( e and f ) Capillary electrophoresis combined with time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis combined with triple quadrupole mass spectrometry (QqQMS) measurements in cation and anion mode were used to generate metabolomics data, which were analyzed using MetaboAnalyst. Of the 116 metabolites identified, enrichment analysis was performed on those with a fold change of ≤ 0.8 ( e ) or ≥ 1.2 ( f ) in the kidneys of luseogliflozin-treated I/R-injured mice (n = 4) relative to those in vehicle-treated I/R-injured mice (n =4). * P < 0.05, ** P < 0.01, as determined by one-way analysis of variance. n.s., not significant.
    Polyclonal Anti Rabbit Pampkα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti‐pampk antibody  (Cell Signaling Technology Inc)
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    Luseogliflozin-mediated enhancement of master regulators of starvation and improvement in mitochondrial function, as shown by kidney metabolomic analysis at 1 week post ischemia/reperfusion (I/R) injury. ( a – d ) Western blot analysis and quantification of kidney expression of <t>pAMPK</t> ( a , b ) and Sirt3 ( c , d ) at 1 week post ischemia/reperfusion (I/R) injury in the following groups of mice: vehicle-treated + sham-operated (n = 6), luseogliflozin-treated + sham-operated (n = 6), vehicle-treated + I/R-injured (n = 6), and luseogliflozin-treated + I/R-injured (n=6). ( e and f ) Capillary electrophoresis combined with time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis combined with triple quadrupole mass spectrometry (QqQMS) measurements in cation and anion mode were used to generate metabolomics data, which were analyzed using MetaboAnalyst. Of the 116 metabolites identified, enrichment analysis was performed on those with a fold change of ≤ 0.8 ( e ) or ≥ 1.2 ( f ) in the kidneys of luseogliflozin-treated I/R-injured mice (n = 4) relative to those in vehicle-treated I/R-injured mice (n =4). * P < 0.05, ** P < 0.01, as determined by one-way analysis of variance. n.s., not significant.
    Rabbit Polyclonal Anti‐Pampk Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti pampkα thr172  (Cell Signaling Technology Inc)
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    rabbit polyclonal pampk  (Abcam)
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    rabbit polyclonal anti pampkα t172  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc rabbit polyclonal anti pampkα t172
    ( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
    Rabbit Polyclonal Anti Pampkα T172, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti pampkα  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc rabbit polyclonal anti pampkα
    ( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
    Rabbit Polyclonal Anti Pampkα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti-pampk polyclonal antibody  (Santa Cruz Biotechnology)
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    ( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
    Rabbit Anti Pampk Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polyclonal pampk  (Cell Signaling Technology Inc)
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    Cell Signaling Technology Inc polyclonal pampk
    ( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
    Polyclonal Pampk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Luseogliflozin-mediated enhancement of master regulators of starvation and improvement in mitochondrial function, as shown by kidney metabolomic analysis at 1 week post ischemia/reperfusion (I/R) injury. ( a – d ) Western blot analysis and quantification of kidney expression of pAMPK ( a , b ) and Sirt3 ( c , d ) at 1 week post ischemia/reperfusion (I/R) injury in the following groups of mice: vehicle-treated + sham-operated (n = 6), luseogliflozin-treated + sham-operated (n = 6), vehicle-treated + I/R-injured (n = 6), and luseogliflozin-treated + I/R-injured (n=6). ( e and f ) Capillary electrophoresis combined with time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis combined with triple quadrupole mass spectrometry (QqQMS) measurements in cation and anion mode were used to generate metabolomics data, which were analyzed using MetaboAnalyst. Of the 116 metabolites identified, enrichment analysis was performed on those with a fold change of ≤ 0.8 ( e ) or ≥ 1.2 ( f ) in the kidneys of luseogliflozin-treated I/R-injured mice (n = 4) relative to those in vehicle-treated I/R-injured mice (n =4). * P < 0.05, ** P < 0.01, as determined by one-way analysis of variance. n.s., not significant.

    Journal: Scientific Reports

    Article Title: SGLT2 inhibition mitigates transition from acute kidney injury to chronic kidney disease by suppressing ferroptosis

    doi: 10.1038/s41598-024-71416-0

    Figure Lengend Snippet: Luseogliflozin-mediated enhancement of master regulators of starvation and improvement in mitochondrial function, as shown by kidney metabolomic analysis at 1 week post ischemia/reperfusion (I/R) injury. ( a – d ) Western blot analysis and quantification of kidney expression of pAMPK ( a , b ) and Sirt3 ( c , d ) at 1 week post ischemia/reperfusion (I/R) injury in the following groups of mice: vehicle-treated + sham-operated (n = 6), luseogliflozin-treated + sham-operated (n = 6), vehicle-treated + I/R-injured (n = 6), and luseogliflozin-treated + I/R-injured (n=6). ( e and f ) Capillary electrophoresis combined with time-of-flight mass spectrometry (CE-TOFMS) and capillary electrophoresis combined with triple quadrupole mass spectrometry (QqQMS) measurements in cation and anion mode were used to generate metabolomics data, which were analyzed using MetaboAnalyst. Of the 116 metabolites identified, enrichment analysis was performed on those with a fold change of ≤ 0.8 ( e ) or ≥ 1.2 ( f ) in the kidneys of luseogliflozin-treated I/R-injured mice (n = 4) relative to those in vehicle-treated I/R-injured mice (n =4). * P < 0.05, ** P < 0.01, as determined by one-way analysis of variance. n.s., not significant.

    Article Snippet: The following primary antibodies were used: monoclonal anti-rabbit α/β-tubulin (1:5000, CST#2148; Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-mouse GAPDH (1:5000, AB8245; Abcam, Cambridge, United Kingdom), monoclonal anti-mouse αSMA (1:1000, ab7817; Abcam, Cambridge, United Kingdom), monoclonal anti- rabbit collagen I, (1:1000, ab21286; Abcam, Cambridge, United Kingdom), monoclonal anti- rabbit fibronectin, (1:1000, ab2413; Abcam, Cambridge, United Kingdom), polyclonal anti-rabbit PPARα (1:1000, sc-9000; Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal anti-rabbit ACADL (1:1000, 17526-1-AP; Proteintech, Rosemont, IL, USA) ,monoclonal anti-mouse CPT1α (1:1000, ab128568; Abcam, Cambridge, United Kingdom), polyclonal anti-rabbit pAMPKα (1:1000, #2531; Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-rabbit SIRT3 (1:1000, #5490S; Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-rabbit PGC1α (1:1000, NOVUSBIO, Centennial, CO, USA) monoclonal anti-rabbit TOM20 (1:1000, #42406; Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-rabbit Nrf2 (1:1000, ab137550; Abcam, Cambridge, United Kingdom), polyclonal anti-rabbit GCLM (1:1000, 14241-1-AP; Proteintech, Rosemont, IL, USA), polyclonal anti-rabbit NQO1 (1:1000, 11451-1-AP; Proteintech, Rosemont, IL, USA), polyclonal anti-rabbit TXNRD1 (1:1000, 11117-1-AP; Proteintech, Rosemont, IL, USA), polyclonal anti-rabbit TfR1 (1:1000, sc-32272; Santa Cruz Biotechnology, Dallas, TX, USA), and monoclonal anti-rabbit Gpx4 (1:1000, 67763-1-Ig; Proteintech, Rosemont, IL, USA).

    Techniques: Western Blot, Expressing, Electrophoresis, Mass Spectrometry, Targeted Proteomics

    Schematic diagram of the therapeutic effect of luseogliflozin, showing how it effectively reduces I/R injury and prevents fibrosis in mice. Under normal conditions, I/R injury reduces tubular mitochondrial function and increases oxidative stress. In addition, tubular metabolism shifts from fatty acid oxidation to glycolysis, resulting in accumulation of polyunsaturated fatty acids (PUFAs) and leading to lipid peroxidation and tubular ferroptosis. Next, macrophage infiltration and inflammatory cytokine production occur, leading to tubular fibrosis and the transition from acute kidney injury (AKI) to chronic kidney disease (CKD). Sodium-glucose cotransporter 2 (SGLT2) inhibitors increase the starvation regulator Sirtuin-3 (Sirt3) and also show a tendency to increase pAMPK. Mitochondrial function is improved, oxidative stress is lowered, and PUFA accumulation is prevented by inhibiting the tubular metabolic shift from fatty acid oxidation to the glycolytic system. Consequently, ferroptosis and development of the AKI to CKD transition are prevented.

    Journal: Scientific Reports

    Article Title: SGLT2 inhibition mitigates transition from acute kidney injury to chronic kidney disease by suppressing ferroptosis

    doi: 10.1038/s41598-024-71416-0

    Figure Lengend Snippet: Schematic diagram of the therapeutic effect of luseogliflozin, showing how it effectively reduces I/R injury and prevents fibrosis in mice. Under normal conditions, I/R injury reduces tubular mitochondrial function and increases oxidative stress. In addition, tubular metabolism shifts from fatty acid oxidation to glycolysis, resulting in accumulation of polyunsaturated fatty acids (PUFAs) and leading to lipid peroxidation and tubular ferroptosis. Next, macrophage infiltration and inflammatory cytokine production occur, leading to tubular fibrosis and the transition from acute kidney injury (AKI) to chronic kidney disease (CKD). Sodium-glucose cotransporter 2 (SGLT2) inhibitors increase the starvation regulator Sirtuin-3 (Sirt3) and also show a tendency to increase pAMPK. Mitochondrial function is improved, oxidative stress is lowered, and PUFA accumulation is prevented by inhibiting the tubular metabolic shift from fatty acid oxidation to the glycolytic system. Consequently, ferroptosis and development of the AKI to CKD transition are prevented.

    Article Snippet: The following primary antibodies were used: monoclonal anti-rabbit α/β-tubulin (1:5000, CST#2148; Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-mouse GAPDH (1:5000, AB8245; Abcam, Cambridge, United Kingdom), monoclonal anti-mouse αSMA (1:1000, ab7817; Abcam, Cambridge, United Kingdom), monoclonal anti- rabbit collagen I, (1:1000, ab21286; Abcam, Cambridge, United Kingdom), monoclonal anti- rabbit fibronectin, (1:1000, ab2413; Abcam, Cambridge, United Kingdom), polyclonal anti-rabbit PPARα (1:1000, sc-9000; Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal anti-rabbit ACADL (1:1000, 17526-1-AP; Proteintech, Rosemont, IL, USA) ,monoclonal anti-mouse CPT1α (1:1000, ab128568; Abcam, Cambridge, United Kingdom), polyclonal anti-rabbit pAMPKα (1:1000, #2531; Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-rabbit SIRT3 (1:1000, #5490S; Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-rabbit PGC1α (1:1000, NOVUSBIO, Centennial, CO, USA) monoclonal anti-rabbit TOM20 (1:1000, #42406; Cell Signaling Technology, Danvers, MA, USA), polyclonal anti-rabbit Nrf2 (1:1000, ab137550; Abcam, Cambridge, United Kingdom), polyclonal anti-rabbit GCLM (1:1000, 14241-1-AP; Proteintech, Rosemont, IL, USA), polyclonal anti-rabbit NQO1 (1:1000, 11451-1-AP; Proteintech, Rosemont, IL, USA), polyclonal anti-rabbit TXNRD1 (1:1000, 11117-1-AP; Proteintech, Rosemont, IL, USA), polyclonal anti-rabbit TfR1 (1:1000, sc-32272; Santa Cruz Biotechnology, Dallas, TX, USA), and monoclonal anti-rabbit Gpx4 (1:1000, 67763-1-Ig; Proteintech, Rosemont, IL, USA).

    Techniques:

    Journal: iScience

    Article Title: Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity

    doi: 10.1016/j.isci.2024.109121

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-pAmpkα (Thr172) , Cell Signaling Technology , #2531.

    Techniques: Enzyme-linked Immunosorbent Assay, Glucose Assay, Sequencing, Software

    ( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of pAMPKα (T172), ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.

    Journal: Science Advances

    Article Title: Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism

    doi: 10.1126/sciadv.abi4797

    Figure Lengend Snippet: ( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of pAMPKα (T172), ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.

    Article Snippet: The following antibodies were used: goat polyclonal anti-actin (I-19) (sc-1616, Santa Cruz Biotechnology), rabbit polyclonal anti-FKBP51 (A301-430A, Bethyl Laboratories), rabbit monoclonal anti-FKBP5 (D5G2, #12210, Cell Signaling Technology), rabbit monoclonal anti-LKB1 (D60C5, #3047, Cell Signaling Technology), rabbit polyclonal anti-pAMPKα T172 (#2531, Cell Signaling Technology), rabbit polyclonal anti-pAMPKα (#2532, Cell Signaling Technology), rabbit polyclonal anti-SKP2 (L70, #4313, Cell Signaling Technology), rabbit anti-pSKP2 S72 (was a gift from Cell Signaling Technology), rabbit polyclonal anti-AKT (#9272, Cell Signaling Technology), rabbit monoclonal anti-pAKT S473 (D9E, #4060, Cell Signaling Technology), rabbit polyclonal anti-p62 (#5114, Cell Signaling Technology), rabbit monoclonal anti-LC3B (D11, #3868, Cell Signaling Technology), rabbit polyclonal anti-pULK1 S757 (#6888, Cell Signaling Technology), rabbit monoclonal anti-pULK1 S555 (D1H4, #5869, Cell Signaling Technology), rabbit monoclonal anti-ULK1 (D8H5, #8054, Cell Signaling Technology), anti-pBECN1 S93/S96 (in mouse S91/S94) (#12476, Cell Signaling Technology), rabbit polyclonal anti-pBECN1 S15 (#84966, Cell Signaling Technology), rabbit polyclonal anti-BECN1 (#3738, Cell Signaling Technology), rabbit polyclonal anti-TSC2 (#3612, Cell Signaling Technology), rabbit polyclonal anti-pTSC2 S1387 (#5584, Cell Signaling Technology), rabbit monoclonal anti-pATG16L1 S278 (EPR19016, ab195242, Abcam), rabbit polyclonal anti-WIPI4 (WDR45) (19194-1-AP, Proteintech), mouse monoclonal anti-WIPI4 (G12, sc-398272, Santa Cruz Biotechnology), rabbit polyclonal anti-WIPI3 (WDR45L) (SAB2102704, Sigma-Aldrich), mouse monoclonal anti-WIPI3 (B-7, sc-514194, Santa Cruz Biotechnology), rabbit polyclonal anti-WIPI2 (#8567, Cell Signaling Technology), rabbit polyclonal anti-WIPI1 (HPA007493, Sigma-Aldrich), rabbit polyclonal anti-AMPKα1 (#2795, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ2 (#2536, Cell Signaling Technology), rabbit polyclonal anti-AMPKα2 (#2757, Cell Signaling Technology), rabbit monoclonal anti-AMPKβ1 (71C10, #4178, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ1 (#4187, Cell Signaling Technology), rabbit polyclonal anti-AMPKβ2 (#4188, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ3 (#2550, Cell Signaling Technology), rabbit monoclonal anti-TSC1 (D43E2, #6935, Cell Signaling Technology), rabbit polyclonal anti-Flag (600-401-383, Rockland Inc.), rabbit polyclonal anti-hypusine (ABS1046, Merck Millipore), rabbit monoclonal anti-eIF5A (D8L8Q, #20765, Cell Signaling Technology), and rabbit polyclonal anti-TFEB (ab245350, Abcam).

    Techniques: Over Expression, Biomarker Discovery, Translocation Assay, Reporter Assay, Control, Comparison

    FKBP51 deletion is depicted in green, and FKBP51 overexpression is depicted in blue. ( A ) Representative blots of autophagy and mTOR markers in FKBP51 MBH-KO mice. ( B ) Quantification of FKBP51 deletion. ( C ) FKBP51 deletion reduced LKB1 and AMPK binding to WIPI4 as well as ( D ) AMPK phosphorylation at T172. ( E ) TSC2-WIPI3 binding was decreased in FKBP51 MBH-KO animals. ( F ) Quantification of mTOR substrate pp70S6K (T389). ( G ) LC3B-II and ( H ) p62 levels in the MBH. ( I ) Representative blots of autophagy and mTOR marker in FKBP51 MBH-OE mice. ( J ) Quantification of viral FKBP51 overexpression. ( K ) FKBP51 overexpression reduced LKB1 and AMPK binding to WIPI4. ( L ) Quantification of AMPK phosphorylation at T172. ( M ) TSC2-WIPI3 binding was decreased. ( N ) Quantification of pp70S6K phosphorylation at T389. ( O ) To assess autophagic flux FKBP51MBH-OE, animals were treated with chloroquine (50 mg/kg), and LC3B-II levels were analyzed 4 hours after treatment. ( P ) FKBP51 overexpression blocked autophagic flux and resulted in an accumulation of p62. ( Q and R ) Quantification of FKBP51, p62, and BECN1, while titrating AAV-HA-FKBP51 virus into mouse neuroblastoma cells. ( S ) MBH FKBP51 regulates autophagy and mTOR signaling in a dose-dependent manner. All data are shown as ±SEM. Data are shown as the relative protein expression compared to control; for (A) to (N), an unpaired Student’s t test was performed. * P < 0.05, ** P < 0.01, and *** P < 0.001.

    Journal: Science Advances

    Article Title: Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism

    doi: 10.1126/sciadv.abi4797

    Figure Lengend Snippet: FKBP51 deletion is depicted in green, and FKBP51 overexpression is depicted in blue. ( A ) Representative blots of autophagy and mTOR markers in FKBP51 MBH-KO mice. ( B ) Quantification of FKBP51 deletion. ( C ) FKBP51 deletion reduced LKB1 and AMPK binding to WIPI4 as well as ( D ) AMPK phosphorylation at T172. ( E ) TSC2-WIPI3 binding was decreased in FKBP51 MBH-KO animals. ( F ) Quantification of mTOR substrate pp70S6K (T389). ( G ) LC3B-II and ( H ) p62 levels in the MBH. ( I ) Representative blots of autophagy and mTOR marker in FKBP51 MBH-OE mice. ( J ) Quantification of viral FKBP51 overexpression. ( K ) FKBP51 overexpression reduced LKB1 and AMPK binding to WIPI4. ( L ) Quantification of AMPK phosphorylation at T172. ( M ) TSC2-WIPI3 binding was decreased. ( N ) Quantification of pp70S6K phosphorylation at T389. ( O ) To assess autophagic flux FKBP51MBH-OE, animals were treated with chloroquine (50 mg/kg), and LC3B-II levels were analyzed 4 hours after treatment. ( P ) FKBP51 overexpression blocked autophagic flux and resulted in an accumulation of p62. ( Q and R ) Quantification of FKBP51, p62, and BECN1, while titrating AAV-HA-FKBP51 virus into mouse neuroblastoma cells. ( S ) MBH FKBP51 regulates autophagy and mTOR signaling in a dose-dependent manner. All data are shown as ±SEM. Data are shown as the relative protein expression compared to control; for (A) to (N), an unpaired Student’s t test was performed. * P < 0.05, ** P < 0.01, and *** P < 0.001.

    Article Snippet: The following antibodies were used: goat polyclonal anti-actin (I-19) (sc-1616, Santa Cruz Biotechnology), rabbit polyclonal anti-FKBP51 (A301-430A, Bethyl Laboratories), rabbit monoclonal anti-FKBP5 (D5G2, #12210, Cell Signaling Technology), rabbit monoclonal anti-LKB1 (D60C5, #3047, Cell Signaling Technology), rabbit polyclonal anti-pAMPKα T172 (#2531, Cell Signaling Technology), rabbit polyclonal anti-pAMPKα (#2532, Cell Signaling Technology), rabbit polyclonal anti-SKP2 (L70, #4313, Cell Signaling Technology), rabbit anti-pSKP2 S72 (was a gift from Cell Signaling Technology), rabbit polyclonal anti-AKT (#9272, Cell Signaling Technology), rabbit monoclonal anti-pAKT S473 (D9E, #4060, Cell Signaling Technology), rabbit polyclonal anti-p62 (#5114, Cell Signaling Technology), rabbit monoclonal anti-LC3B (D11, #3868, Cell Signaling Technology), rabbit polyclonal anti-pULK1 S757 (#6888, Cell Signaling Technology), rabbit monoclonal anti-pULK1 S555 (D1H4, #5869, Cell Signaling Technology), rabbit monoclonal anti-ULK1 (D8H5, #8054, Cell Signaling Technology), anti-pBECN1 S93/S96 (in mouse S91/S94) (#12476, Cell Signaling Technology), rabbit polyclonal anti-pBECN1 S15 (#84966, Cell Signaling Technology), rabbit polyclonal anti-BECN1 (#3738, Cell Signaling Technology), rabbit polyclonal anti-TSC2 (#3612, Cell Signaling Technology), rabbit polyclonal anti-pTSC2 S1387 (#5584, Cell Signaling Technology), rabbit monoclonal anti-pATG16L1 S278 (EPR19016, ab195242, Abcam), rabbit polyclonal anti-WIPI4 (WDR45) (19194-1-AP, Proteintech), mouse monoclonal anti-WIPI4 (G12, sc-398272, Santa Cruz Biotechnology), rabbit polyclonal anti-WIPI3 (WDR45L) (SAB2102704, Sigma-Aldrich), mouse monoclonal anti-WIPI3 (B-7, sc-514194, Santa Cruz Biotechnology), rabbit polyclonal anti-WIPI2 (#8567, Cell Signaling Technology), rabbit polyclonal anti-WIPI1 (HPA007493, Sigma-Aldrich), rabbit polyclonal anti-AMPKα1 (#2795, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ2 (#2536, Cell Signaling Technology), rabbit polyclonal anti-AMPKα2 (#2757, Cell Signaling Technology), rabbit monoclonal anti-AMPKβ1 (71C10, #4178, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ1 (#4187, Cell Signaling Technology), rabbit polyclonal anti-AMPKβ2 (#4188, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ3 (#2550, Cell Signaling Technology), rabbit monoclonal anti-TSC1 (D43E2, #6935, Cell Signaling Technology), rabbit polyclonal anti-Flag (600-401-383, Rockland Inc.), rabbit polyclonal anti-hypusine (ABS1046, Merck Millipore), rabbit monoclonal anti-eIF5A (D8L8Q, #20765, Cell Signaling Technology), and rabbit polyclonal anti-TFEB (ab245350, Abcam).

    Techniques: Over Expression, Binding Assay, Phospho-proteomics, Marker, Virus, Expressing, Control

    FKBP51 overexpression is depicted in blue, and FKBP51 deletion is depicted in green. ( A and B ) Representative decrease in tissue NE content after α-MPT injection (left) and turnover rate (right) were determined on SM and eWAT (see fig. S8 for pancreas, heart, iWAT, and BAT tissues). Quantification of ( C ) pAMPK (T172) and ( D ) pp70S6K (T389), and ( E ) p62 level in the SM and eWAT. ( F ) Representative blots. ( G to H ) FKBP51 overexpression increased autophagic flux and in SM and eWAT. ( I ) Representative blots of chloroquine the experiment. Quantification of ( J ) pAMPK (T172), ( K ) pp70S6K (T389), ( L ) LC3B-II, and ( M ) p62 levels in SM and eWAT in animals lacking FKBP51 in the MBH. ( N ) Representative blots of FKBP51 MBH-KO protein analysis. All data are shown as ±SEM. Protein data are shown as the relative protein expression compared to control. A two-way ANOVA was performed, followed by a Tukey’s multiple comparison test in (F) and (G). For (A) to (E) and (I) to (L), an unpaired Student’s t test was performed. * P < 0.05, ** P < 0.01, and *** P < 0.001.

    Journal: Science Advances

    Article Title: Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism

    doi: 10.1126/sciadv.abi4797

    Figure Lengend Snippet: FKBP51 overexpression is depicted in blue, and FKBP51 deletion is depicted in green. ( A and B ) Representative decrease in tissue NE content after α-MPT injection (left) and turnover rate (right) were determined on SM and eWAT (see fig. S8 for pancreas, heart, iWAT, and BAT tissues). Quantification of ( C ) pAMPK (T172) and ( D ) pp70S6K (T389), and ( E ) p62 level in the SM and eWAT. ( F ) Representative blots. ( G to H ) FKBP51 overexpression increased autophagic flux and in SM and eWAT. ( I ) Representative blots of chloroquine the experiment. Quantification of ( J ) pAMPK (T172), ( K ) pp70S6K (T389), ( L ) LC3B-II, and ( M ) p62 levels in SM and eWAT in animals lacking FKBP51 in the MBH. ( N ) Representative blots of FKBP51 MBH-KO protein analysis. All data are shown as ±SEM. Protein data are shown as the relative protein expression compared to control. A two-way ANOVA was performed, followed by a Tukey’s multiple comparison test in (F) and (G). For (A) to (E) and (I) to (L), an unpaired Student’s t test was performed. * P < 0.05, ** P < 0.01, and *** P < 0.001.

    Article Snippet: The following antibodies were used: goat polyclonal anti-actin (I-19) (sc-1616, Santa Cruz Biotechnology), rabbit polyclonal anti-FKBP51 (A301-430A, Bethyl Laboratories), rabbit monoclonal anti-FKBP5 (D5G2, #12210, Cell Signaling Technology), rabbit monoclonal anti-LKB1 (D60C5, #3047, Cell Signaling Technology), rabbit polyclonal anti-pAMPKα T172 (#2531, Cell Signaling Technology), rabbit polyclonal anti-pAMPKα (#2532, Cell Signaling Technology), rabbit polyclonal anti-SKP2 (L70, #4313, Cell Signaling Technology), rabbit anti-pSKP2 S72 (was a gift from Cell Signaling Technology), rabbit polyclonal anti-AKT (#9272, Cell Signaling Technology), rabbit monoclonal anti-pAKT S473 (D9E, #4060, Cell Signaling Technology), rabbit polyclonal anti-p62 (#5114, Cell Signaling Technology), rabbit monoclonal anti-LC3B (D11, #3868, Cell Signaling Technology), rabbit polyclonal anti-pULK1 S757 (#6888, Cell Signaling Technology), rabbit monoclonal anti-pULK1 S555 (D1H4, #5869, Cell Signaling Technology), rabbit monoclonal anti-ULK1 (D8H5, #8054, Cell Signaling Technology), anti-pBECN1 S93/S96 (in mouse S91/S94) (#12476, Cell Signaling Technology), rabbit polyclonal anti-pBECN1 S15 (#84966, Cell Signaling Technology), rabbit polyclonal anti-BECN1 (#3738, Cell Signaling Technology), rabbit polyclonal anti-TSC2 (#3612, Cell Signaling Technology), rabbit polyclonal anti-pTSC2 S1387 (#5584, Cell Signaling Technology), rabbit monoclonal anti-pATG16L1 S278 (EPR19016, ab195242, Abcam), rabbit polyclonal anti-WIPI4 (WDR45) (19194-1-AP, Proteintech), mouse monoclonal anti-WIPI4 (G12, sc-398272, Santa Cruz Biotechnology), rabbit polyclonal anti-WIPI3 (WDR45L) (SAB2102704, Sigma-Aldrich), mouse monoclonal anti-WIPI3 (B-7, sc-514194, Santa Cruz Biotechnology), rabbit polyclonal anti-WIPI2 (#8567, Cell Signaling Technology), rabbit polyclonal anti-WIPI1 (HPA007493, Sigma-Aldrich), rabbit polyclonal anti-AMPKα1 (#2795, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ2 (#2536, Cell Signaling Technology), rabbit polyclonal anti-AMPKα2 (#2757, Cell Signaling Technology), rabbit monoclonal anti-AMPKβ1 (71C10, #4178, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ1 (#4187, Cell Signaling Technology), rabbit polyclonal anti-AMPKβ2 (#4188, Cell Signaling Technology), rabbit polyclonal anti-AMPKγ3 (#2550, Cell Signaling Technology), rabbit monoclonal anti-TSC1 (D43E2, #6935, Cell Signaling Technology), rabbit polyclonal anti-Flag (600-401-383, Rockland Inc.), rabbit polyclonal anti-hypusine (ABS1046, Merck Millipore), rabbit monoclonal anti-eIF5A (D8L8Q, #20765, Cell Signaling Technology), and rabbit polyclonal anti-TFEB (ab245350, Abcam).

    Techniques: Over Expression, Injection, Expressing, Control, Comparison