rabbit polyclonal antibodies targeting pampk thr172 cat Search Results


gsk3b  (Cell Signaling Technology Inc)
99
Cell Signaling Technology Inc gsk3b
Fig. 1 | AdipoRon clears phosphorylated tau and NFT via AMPK and <t>GSK3b.</t> A Immunofluorescence of phosphorylated Tau (pTau) at S202/T205 and NFT (MC-1 antibody) in Tau neurons following 24-h treatment with 10 μM AdipoRon ± 8 μM AMPK inhibitor Compound C (pTau: n = 6 mice, n = 43–58 neurons per treatment; NFT: n = 8 mice, n = 53–79 neurons per treatment), quantification of immunofluorescence images above; one-way ANOVA with Tukey’s multiple comparison’s test, scale bar 15 μm. B Immunofluorescence detection and quantifica- tion of phosphorylated AMPK and total AMPK in individual Tau neurons following 10-min treatment with 10 μM AdipoRon (pAMPK: n = 5 mice, n = 57–63 neurons per treatment; total AMPK: n = 3 mice, n = 44–56 neurons per treatment); Student’s t-test with Welch’s correction. C Immunofluorescence detection and quantifica- tion of phosphorylated GSK3b and total GSK3b in Tau neurons following 10-min treatment with 10 μM AdipoRon (pGSK3b: n = 5 mice, n = 42–47 neurons per treatment; total GSK3b: n = 5 mice, n = 37–55 neurons per treatment), Student’s t-test with Welch’s correction. D Immunofluorescence detection of pTau after 24-h treatment with 15 mM LiCl (n = 6 mice; n = 40–43 neurons per treatment). Data shown as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Gsk3b, 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 polyclonal anti pampkα t172  (Cell Signaling Technology Inc)
97
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; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
Rabbit Polyclonal Anti Pampkα T172, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit monoclonal pampk thr172  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc rabbit monoclonal pampk thr172
( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ 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 Monoclonal Pampk Thr172, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pampk  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc 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; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
Pampk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pampk thr172  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc pampk thr172
( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ 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.
Pampk Thr172, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pampka  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc pampka
( 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; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
Pampka, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit phosphorylated ampkα  (Cell Signaling Technology Inc)
99
Cell Signaling Technology Inc rabbit phosphorylated ampkα
( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ 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 Phosphorylated Ampkα, 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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ab63473 rabbit anti pampk thr172 pe bioss  (Bioss)
86
Bioss ab63473 rabbit anti pampk thr172 pe bioss
( 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; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
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rabbit antibodies against ampk  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc rabbit antibodies against ampk
( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ 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.
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rabbit anti pampk monoclonal antibody  (Cell Signaling Technology Inc)
98
Cell Signaling Technology Inc rabbit anti pampk monoclonal antibody
( 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; $$ P < 0.01. Two-way ANOVA was performed in (A) to (C) and followed by a Tukey’s multiple comparisons test. One-way ANOVA was performed for (I) and (L), followed by a Dunnett’s multiple comparison test. The unpaired Student’s t test was performed for (E) to (G). *, significant genotype effect; $, significant starvation effect; #, significant treatment effect.
Rabbit Anti Pampk Monoclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit pampk  (Cell Signaling Technology Inc)
97
Cell Signaling Technology Inc rabbit pampk
( A ) WT or FKBP51 KO cells were starved in HBSS medium for 4 hours to induce autophagy, followed by quantification of <t>pAMPKα</t> <t>(T172),</t> ( B ) p62, and ( C ) pp70S6K (T389). Representative blots are shown in ( D ). FKBP51 overexpression (FKBP51 OE) in N2a cells (see fig. S3D for validation) enhanced autophagy signaling. Quantification of ( E ) pAMPKα (T172), ( F ) pp70S6K (T389), ( G ) p62, and ( H ) representative blots. ( I ) Quantification of autophagic flux in FKBP51 KO and FKBP51 OE cells in response to starvation. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( J ) Representative blots of autophagic flux measurements. ( K ) Representative pictures of TFEB nuclear localization/translocation. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 10 μm. ( L ) Quantification of TFEB reporter assay. BL, baseline. All data (A to J) are shown as relative fold change compared to control condition; ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001; ## P < 0.01, ### P < 0.001; $$ 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.
Rabbit Pampk, 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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pampkα thr172  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc pampkα thr172
( 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.
Pampkα Thr172, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Fig. 1 | AdipoRon clears phosphorylated tau and NFT via AMPK and GSK3b. A Immunofluorescence of phosphorylated Tau (pTau) at S202/T205 and NFT (MC-1 antibody) in Tau neurons following 24-h treatment with 10 μM AdipoRon ± 8 μM AMPK inhibitor Compound C (pTau: n = 6 mice, n = 43–58 neurons per treatment; NFT: n = 8 mice, n = 53–79 neurons per treatment), quantification of immunofluorescence images above; one-way ANOVA with Tukey’s multiple comparison’s test, scale bar 15 μm. B Immunofluorescence detection and quantifica- tion of phosphorylated AMPK and total AMPK in individual Tau neurons following 10-min treatment with 10 μM AdipoRon (pAMPK: n = 5 mice, n = 57–63 neurons per treatment; total AMPK: n = 3 mice, n = 44–56 neurons per treatment); Student’s t-test with Welch’s correction. C Immunofluorescence detection and quantifica- tion of phosphorylated GSK3b and total GSK3b in Tau neurons following 10-min treatment with 10 μM AdipoRon (pGSK3b: n = 5 mice, n = 42–47 neurons per treatment; total GSK3b: n = 5 mice, n = 37–55 neurons per treatment), Student’s t-test with Welch’s correction. D Immunofluorescence detection of pTau after 24-h treatment with 15 mM LiCl (n = 6 mice; n = 40–43 neurons per treatment). Data shown as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Journal: Communications biology

Article Title: Reversal of neuronal tau pathology via adiponectin receptor activation.

doi: 10.1038/s42003-024-07391-z

Figure Lengend Snippet: Fig. 1 | AdipoRon clears phosphorylated tau and NFT via AMPK and GSK3b. A Immunofluorescence of phosphorylated Tau (pTau) at S202/T205 and NFT (MC-1 antibody) in Tau neurons following 24-h treatment with 10 μM AdipoRon ± 8 μM AMPK inhibitor Compound C (pTau: n = 6 mice, n = 43–58 neurons per treatment; NFT: n = 8 mice, n = 53–79 neurons per treatment), quantification of immunofluorescence images above; one-way ANOVA with Tukey’s multiple comparison’s test, scale bar 15 μm. B Immunofluorescence detection and quantifica- tion of phosphorylated AMPK and total AMPK in individual Tau neurons following 10-min treatment with 10 μM AdipoRon (pAMPK: n = 5 mice, n = 57–63 neurons per treatment; total AMPK: n = 3 mice, n = 44–56 neurons per treatment); Student’s t-test with Welch’s correction. C Immunofluorescence detection and quantifica- tion of phosphorylated GSK3b and total GSK3b in Tau neurons following 10-min treatment with 10 μM AdipoRon (pGSK3b: n = 5 mice, n = 42–47 neurons per treatment; total GSK3b: n = 5 mice, n = 37–55 neurons per treatment), Student’s t-test with Welch’s correction. D Immunofluorescence detection of pTau after 24-h treatment with 15 mM LiCl (n = 6 mice; n = 40–43 neurons per treatment). Data shown as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Primary antibodies used: MAP2 (ab5392; Abcam), AT-8 (MN1020; Thermo Scientific), MC-1 (Peter Davies lab), LC3b (ab48394; Abcam), p62 (23214; Cell Signaling Technologies), pGSK3b (Ser9) (9336; Cell Signaling Technologies), GSK3b (9832; Cell Signaling Technologies, pAMPK (Thr172) (2535; Cell Signaling Technologies), AMPK (2532; Cell Signaling Technologies), Tomm20 (ab56783; Abcam), pJNK (Thr183/ Tyr185 (9255; Cell Signaling Technologies), and JNK(9252; Cell Signaling Technologies).

Techniques:

( 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