Anti p ampk t172

Cell culture reagents were purchased from Invitrogen (Carlsbad, CA); commerciall\y available Astaxanthin (AX) powder was purchased from Fuji Chemical Industries USA, Inc. (P2AF, Burlington, NJ); anti‐Akt, anti‐p‐Akt (S473), anti‐AMPK, anti‐p‐AMPK (T172), anti‐p‐Acetyl‐CoA carboxylase (ACC), anti‐ACC, and anti‐β‐Actin were purchased from Cell Signaling Technology (Danvers, MA); anti‐Sirt1 was purchased from Millipore (Billerica MA). Anti‐PGC‐1 and total OXPHOS rodent WB antibody cocktail were purchased from Abcam (Cambridge, MA). 5‐Aminoimidazole‐4‐carboxamide ribonucleotide (AICAR), AMPK agonist, was purchased from AdipoGen Life Science (AG‐CR1‐0061‐M050). Horse radish peroxidase‐conjugated antirat or antirabbit secondary antibody and the ECL western blot detection reagent were obtained from GE Healthcare (Buckinghamshire, UK). All other reagents were purchased from Sigma‐Aldrich.

Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco (Rockville, MD, USA). Penicillin-streptomycin solution was purchased from Hyclone (Provo, UT, USA). Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3T3-L1 preadipocytes were purchased from ATCC (Manassas, VA, USA). Bilobalide was obtained from the National Institutes for Food and Drug Control (Beijing, China). Primary antibodies specific for anti-pACC1 (S79), anti-ACC1, anti-FASN, anti-perilipin A, anti-ATGL, anti-C/EBPα, anti-PPARγ, anti-HSL, anti-pHSL (S563), anti-GLUT-4, anti-CPT-1α, anti-AMPK, and anti-pAMPK (T172) were acquired from Cell Signaling Technology (Danvers, MD, USA). Anti-SREBP-1c was acquired from Abcam (Cambridge, UK). Anti-β-Actin and goat anti-mouse and goat anti-rabbit IgG secondary antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). The AMPK assay kit was purchased from Cell Signaling Technology.

Media, sera and antibiotics for cell culture were from Lonza (Walkersville, MD, USA). Protein electrophoresis and western blot reagents were from Bio-Rad (Richmond, VA, USA) and electrochemiluminescence reagents from Pierce (Rockford, IL, USA). Insulin was from Eli Lilly (Florence, Italy). The antibodies used were: anti-GLP-1 receptor (kind gift of Drs. Wanda Dolci and Bernard Thorens [10 (link), 23 (link)], anti-Insulin receptor (IR), anti-p-tyrosine (Biosource, Camarillo, CA), anti-IRS-1 (Millipore, Billerica, MA, USA), anti-p85 subunit of PI3-kinase, anti-Akt, anti-p-Akt S473, anti-p-AMPK T172, anti-AMPK, anti-p-ACC S78/80, anti-ACC, anti-p-JNK T183/Y185, anti-JNK, anti-p-AS160T642, anti-AS160 (Cell Signaling Technology, Beverly, MA, USA), anti-ERK1/2, anti-p-ERK1/2 (Santa Cruz, CA, USA), anti-GLUT4 antibodies [24 (link)], and anti-αTubulin (Sigma, St Louis, MO, USA). 5-amino-4-imidazole carboxamide riboside (AICAR), Compound C, and LY294002 were from Calbiochem (La Jolla, CA). SiRNAs were from Riboxx (Radebeul, Germany). Methylglyoxal (MGO) was from SIGMA-Aldrich (St Louis, MO, USA). Exenatide and liraglutide were kind gifts from Amylin and Novo Nordisk, respectively.

Human breast cancer cell lines MDA-MB-468, BT474, SKBR-3 and normal breast epithelial cell line Hs578Bs were purchased from the Cell Center of Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human breast cancer cell lines MCF-7, MDA-MB-231 and human embryonic kidney cell line HEK293T were kept in our laboratory. EBSS (Hyclone) starvation was carried out to switch the culture medium from the complete medium to EBSS medium. Commercially available antibodies and dilutions used are as follows: anti-ITM2A (Proteintech, cat: 18306–1-AP, 1:1000 dilution), anti-GAPDH (Proteintech, cat: 60004–1-Ig, 1:1000 dilution), anti-LC3 (Cell Signaling Technology, cat: 12741, 1:1000 dilution), anti-P62/SQSTM1 (Boster, cat: BM4385, 1:1000 dilution), anti-Rabbit Flag (EMD Millipore, cat: PM020A, 1:1000 dilution), anti-pAMPK-T172 (Cell Signaling Technology, cat: 2535, 1:1000 dilution), anti-AMPK (Boster, cat: A30453, 1:500 dilution), anti-p4EBP1-T37/46 (Cell Signaling Technology, cat: 2855, 1:1000 dilution), anti-4EBP1 (Proteintech, cat: 60246–1-Ig, 1:1000 dilution). anti-phospho-MBP (EMD Millipore, cat: 05–429, 1:1000 dilution), anti-MBP (Proteintech, cat: 10458–1-AP, 1:1000 dilution), anti-HUNK (Invitrogen, cat: PA5–28765, 1:1000 dilution).

The primary antibodies that included anti-LC3 (#12741), anti-p-AMPK (T172) (# 2535), anti-AMPK (#2532), anti-p-ULK1 (Ser317) (# 12753), anti-ULK1 (# 6439), anti-β-Actin (# 4970) and goat anti-rabbit (# 7074) and goat anti-mouse (# 7076) HRP-conjugated secondary antibodies were all obtained from Cell Signaling Technology (Beverly, Massachusetts, USA). The monoclonal antibody of NNMT was prepared in our lab as previously described [15 (link)]. The H₂O₂ solution was obtained from Sigma (#H1009).