Journal: ACS Pharmacology & Translational Science

Article Title: Molecular Signature for Receptor Engagement in the Metabolic Peptide Hormone Amylin

doi: 10.1021/acsptsci.8b00002

Figure Lengend Snippet: (a) Receptor subunit composition, (b) activation of signaling pathways at the corresponding receptors by human amylin (hAMY), (c) activation of signaling pathways at the corresponding receptors by all peptides. In panel b, the concentration response curves are the combined mean data from four or five independent experiments (cAMP, pCREB n = 5, IP1, pERK1/2 n = 4). In panel c, potency data are summarized in radial plots showing mean pEC50 values from between three and five individual experiments. Exact experimental n is provided in Tables SB1–4. All errors are s.e.m. pERK1/2 data is the 15 min time point.

Article Snippet: Phosphorylated (p) extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB were detected using the AlphaLISA SureFire Ultra pERK1/2 (Thr202/Tyr204) or the AlphaLISA SureFire Ultra pCREB (Ser133) assay kits (PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) as per the manufacturer’s protocol.

Techniques: Activation Assay, Concentration Assay

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: International Journal of Molecular Sciences

Article Title: COX-2/sEH Dual Inhibitor Alleviates Hepatocyte Senescence in NAFLD Mice by Restoring Autophagy through Sirt1/PI3K/AKT/mTOR

doi: 10.3390/ijms23158267

Figure Lengend Snippet: PTUPB attenuates hepatocyte senescence by enhancing autophagy in AML12 cells. The fluorescence intensity of liver LC3 was detected by immunofluorescence (( A ), scale bars = 50 μm, n = 6). Image J was used to analyze the fluorescence intensity of LC3 (( B ), n = 6). Cells were treated with PTUPB (1 μM) for 1 h before treatment with PA (200 µM). The protein expressions of p62, LC3I/II, and Fundc1 were measured by Western blot and quantitatively analyzed in AML12 cells treated with PA for 48 h (( C – F ), n = 3). Cells were treated with PTUPB (1 μM) and 3-MA (5 mM) for 1 h before treatment with PA (200 µM). The protein expressions of p16, γH2AX, Fundc1, LC3II, and p62 were measured by Western blot and quantitatively analyzed in AML12 cells (( G – L ), n = 3). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey’s test was used as a post hoc test for pairwise comparisons. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Anti-p62 polyclonal antibody , Immunoway , YT7058.

Techniques: Fluorescence, Immunofluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: COX-2/sEH Dual Inhibitor Alleviates Hepatocyte Senescence in NAFLD Mice by Restoring Autophagy through Sirt1/PI3K/AKT/mTOR

doi: 10.3390/ijms23158267

Figure Lengend Snippet: Antibodies were used in this study.

Article Snippet: Anti-p62 polyclonal antibody , Immunoway , YT7058.

Techniques: