Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing direct interaction between AMPKγ1 and ULK1. B. Evolutionally conserved sequence alignment of AMPKγ1 in mammals. C. Immunoblot of proteins subjected to a kinase assay using purified trimeric AMPK consisting of AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A mutant with and without ULK1 showing direct phosphorylation of AMPKγ1 at S260/T262 by ULK1. D. Immunoblots of proteins subjected to a kinase assay using purified monomeric AMPKγ1 and ULK1. E. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing AMPKγ1 bound to T-ULK1 is likely to be phosphorylated at S260/T262.

Article Snippet: The AMPKγ1 mutant vector wherein Ser260 and Thr262 were replaced with alanine was created using parent-template backbone mouse AMPKγ1- full-length vector (# 15996, Addgene) and primers with the desired mutation.

Techniques: Immunoprecipitation, Expressing, Sequencing, Western Blot, Kinase Assay, Purification, Mutagenesis, Phospho-proteomics

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 with/without Halo-tagged ULK1 expression in HEK293T cells showing AMPK bound to T-ULK1 is likely to be phosphorylated at P-AMPKα Thr172 . Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression when ULK1 is overexpressed (n = 3). A. B. Immunoprecipitation of FLAG-tagged AMPKγ1 WT or AMPKγ1 S260A/T262A with Halo- tagged ULK1 expression in HEK293T cells. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A when ULK1 is overexpressed (n = 4). B. Immunoprecipitation of FLAG-tagged AMPKγ1 with Halo-tagged ULK1 expression in HEK293T cells treated with/without BL918 (selective ULK1 activator) for 15 minutes. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 (lower) and P-AMPKα Thr172 (upper) expression (n = 3). C. Immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A with/without 15 minutes BL918 (selective ULK1 activator, 5 μM) treatment (n = 3). D. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with/without BL918 for 15 minutes (n = 3). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: The AMPKγ1 mutant vector wherein Ser260 and Thr262 were replaced with alanine was created using parent-template backbone mouse AMPKγ1- full-length vector (# 15996, Addgene) and primers with the desired mutation.

Techniques: Immunoprecipitation, Expressing, Western Blot, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 8). B. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in livers of WT or Ulk1 −/− mice following starvation (n = 6). C. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 expression in kidneys of mice subjected to sham operation or 5/6 nephrectomy to induce chronic kidney disease (CKD) (n = 8). P values ( * P < 0.05, ** P < 0.01) were calculated using a two-tailed unpaired t -test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: The AMPKγ1 mutant vector wherein Ser260 and Thr262 were replaced with alanine was created using parent-template backbone mouse AMPKγ1- full-length vector (# 15996, Addgene) and primers with the desired mutation.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A, B. Representative immunoblots (A) and densitometric analysis (B) evaluating the sensitivity of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or S260A/T262A mutant to 12h-AICAR treatment (n = 3). A. C. Crystal structure revealing the role of Ser260 and Thr262 in maintaining the structure of AMPKγ1. B. D. Fluorescence intensity of Mant-AMP (fluorescent dye) bound to purified trimeric AMPK protein consisting of AMPKγ1 WT or AMPKγ1 S260A/T262A with/without ULK1 (n = 5) P values ( * P < 0.05) were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: The AMPKγ1 mutant vector wherein Ser260 and Thr262 were replaced with alanine was created using parent-template backbone mouse AMPKγ1- full-length vector (# 15996, Addgene) and primers with the desired mutation.

Techniques: Western Blot, Mutagenesis, Fluorescence, Purification

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A treated with/without MK8722 (n = 3). B. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without MK8722 treatment (n = 3). C. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without MK8722 at 30 mg/kg BW (n = 5). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or a one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: The AMPKγ1 mutant vector wherein Ser260 and Thr262 were replaced with alanine was created using parent-template backbone mouse AMPKγ1- full-length vector (# 15996, Addgene) and primers with the desired mutation.

Techniques: Western Blot, Expressing, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing direct interaction between AMPKγ1 and ULK1. B. Evolutionally conserved sequence alignment of AMPKγ1 in mammals. C. Immunoblot of proteins subjected to a kinase assay using purified trimeric AMPK consisting of AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A mutant with and without ULK1 showing direct phosphorylation of AMPKγ1 at S260/T262 by ULK1. D. Immunoblots of proteins subjected to a kinase assay using purified monomeric AMPKγ1 and ULK1. E. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing AMPKγ1 bound to T-ULK1 is likely to be phosphorylated at S260/T262.

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Immunoprecipitation, Expressing, Sequencing, Western Blot, Kinase Assay, Purification, Mutagenesis, Phospho-proteomics

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 with/without Halo-tagged ULK1 expression in HEK293T cells showing AMPK bound to T-ULK1 is likely to be phosphorylated at P-AMPKα Thr172 . Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression when ULK1 is overexpressed (n = 3). A. B. Immunoprecipitation of FLAG-tagged AMPKγ1 WT or AMPKγ1 S260A/T262A with Halo- tagged ULK1 expression in HEK293T cells. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A when ULK1 is overexpressed (n = 4). B. Immunoprecipitation of FLAG-tagged AMPKγ1 with Halo-tagged ULK1 expression in HEK293T cells treated with/without BL918 (selective ULK1 activator) for 15 minutes. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 (lower) and P-AMPKα Thr172 (upper) expression (n = 3). C. Immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A with/without 15 minutes BL918 (selective ULK1 activator, 5 μM) treatment (n = 3). D. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with/without BL918 for 15 minutes (n = 3). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Immunoprecipitation, Expressing, Western Blot, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 8). B. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in livers of WT or Ulk1 −/− mice following starvation (n = 6). C. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 expression in kidneys of mice subjected to sham operation or 5/6 nephrectomy to induce chronic kidney disease (CKD) (n = 8). P values ( * P < 0.05, ** P < 0.01) were calculated using a two-tailed unpaired t -test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A, B. Representative immunoblots (A) and densitometric analysis (B) evaluating the sensitivity of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or S260A/T262A mutant to 12h-AICAR treatment (n = 3). A. C. Crystal structure revealing the role of Ser260 and Thr262 in maintaining the structure of AMPKγ1. B. D. Fluorescence intensity of Mant-AMP (fluorescent dye) bound to purified trimeric AMPK protein consisting of AMPKγ1 WT or AMPKγ1 S260A/T262A with/without ULK1 (n = 5) P values ( * P < 0.05) were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Western Blot, Mutagenesis, Fluorescence, Purification

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A treated with/without MK8722 (n = 3). B. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without MK8722 treatment (n = 3). C. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without MK8722 at 30 mg/kg BW (n = 5). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or a one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Western Blot, Expressing, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing direct interaction between AMPKγ1 and ULK1. B. Evolutionally conserved sequence alignment of AMPKγ1 in mammals. C. Immunoblot of proteins subjected to a kinase assay using purified trimeric AMPK consisting of AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A mutant with and without ULK1 showing direct phosphorylation of AMPKγ1 at S260/T262 by ULK1. D. Immunoblots of proteins subjected to a kinase assay using purified monomeric AMPKγ1 and ULK1. E. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing AMPKγ1 bound to T-ULK1 is likely to be phosphorylated at S260/T262.

Article Snippet: Polymerase chain reaction (PCR) was used to isolate mouse AMPKγ1 cDNA from a mouse AMPKγ1-HA vector (#40605, Addgene, Watertown, MA, USA).

Techniques: Immunoprecipitation, Expressing, Sequencing, Western Blot, Kinase Assay, Purification, Mutagenesis

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 with/without Halo-tagged ULK1 expression in HEK293T cells showing AMPK bound to T-ULK1 is likely to be phosphorylated at P-AMPKα Thr172 . Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression when ULK1 is overexpressed (n = 3). A. B. Immunoprecipitation of FLAG-tagged AMPKγ1 WT or AMPKγ1 S260A/T262A with Halo- tagged ULK1 expression in HEK293T cells. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A when ULK1 is overexpressed (n = 4). B. Immunoprecipitation of FLAG-tagged AMPKγ1 with Halo-tagged ULK1 expression in HEK293T cells treated with/without BL918 (selective ULK1 activator) for 15 minutes. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 (lower) and P-AMPKα Thr172 (upper) expression (n = 3). C. Immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A with/without 15 minutes BL918 (selective ULK1 activator, 5 μM) treatment (n = 3). D. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with/without BL918 for 15 minutes (n = 3). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: Polymerase chain reaction (PCR) was used to isolate mouse AMPKγ1 cDNA from a mouse AMPKγ1-HA vector (#40605, Addgene, Watertown, MA, USA).

Techniques: Immunoprecipitation, Expressing, Western Blot, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 8). B. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in livers of WT or Ulk1 −/− mice following starvation (n = 6). C. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 expression in kidneys of mice subjected to sham operation or 5/6 nephrectomy to induce chronic kidney disease (CKD) (n = 8). P values ( * P < 0.05, ** P < 0.01) were calculated using a two-tailed unpaired t -test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: Polymerase chain reaction (PCR) was used to isolate mouse AMPKγ1 cDNA from a mouse AMPKγ1-HA vector (#40605, Addgene, Watertown, MA, USA).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A, B. Representative immunoblots (A) and densitometric analysis (B) evaluating the sensitivity of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or S260A/T262A mutant to 12h-AICAR treatment (n = 3). A. C. Crystal structure revealing the role of Ser260 and Thr262 in maintaining the structure of AMPKγ1. B. D. Fluorescence intensity of Mant-AMP (fluorescent dye) bound to purified trimeric AMPK protein consisting of AMPKγ1 WT or AMPKγ1 S260A/T262A with/without ULK1 (n = 5) P values ( * P < 0.05) were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: Polymerase chain reaction (PCR) was used to isolate mouse AMPKγ1 cDNA from a mouse AMPKγ1-HA vector (#40605, Addgene, Watertown, MA, USA).

Techniques: Western Blot, Mutagenesis, Fluorescence, Purification

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A treated with/without MK8722 (n = 3). B. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without MK8722 treatment (n = 3). C. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without MK8722 at 30 mg/kg BW (n = 5). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or a one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: Polymerase chain reaction (PCR) was used to isolate mouse AMPKγ1 cDNA from a mouse AMPKγ1-HA vector (#40605, Addgene, Watertown, MA, USA).

Techniques: Western Blot, Expressing, Transfection, Two Tailed Test