Anti ulk1

DMEM (Gibco, C11965500BT), fetal bovine serum (Gibco, 16000044), EBSS (MACGENE, CC026), FITC Annexin V apoptosis detection kit I (BD Biosciences, 556547), cell counting kit (YEASEN, QF0026), lysis buffer (Beyotime, P0013), PVDF membranes (Millipore, ISEQ00010), albumin bovine V (Solarbio, A8020), lipofectamine 2000 (Invitrogen, 11668027), Dual-Luciferase^® Reporter Assay System (Promega, E1910), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining kit (Promega, G3250). Bafilomycin A1 was purchased from Sigma. The following antibodies were used: anti-ULK1 (Sigma-Aldrich, A7481), anti-P62 (MBL, PM045), anti-P62 (Abcam, ab56416), anti-LC3B (Sigma, L7543), anti-c-Myc monoclonal antibody (Sigma, C3956), anti-β-actin (Sino Biological, 100162-RP02-100), anti-Gapdh (Transgen, HC301), anti-ULK1 (Sigma, A7481), anti-ATG5 (Sigma, A0731), anti-ATG7 (Sigma, A2856), anti-Beclin1 (CST, 3738), anti-Tom20 (BD Biosciences, 612278), DAPI (CST, 4083), HRP Affinipure goat anti-mouse IgG (Earthox, E030110), and HRP Affinipure goat anti-rabbit IgG (Earthox, E030120). The following fluorescent secondary antibodies were used: Alexa Fluor 555-labeled donkey anti-mouse IgG antibodies (Invitrogen, A31570) and Alexa Fluor 488-labeled donkey anti-rabbit IgG antibodies (Invitrogen, A21206).

ESCs were either treated or not treated with 100 μM Chloroquine (Sigma) for 4 h. After the treatments, the cells were collected and lysed by RIPA buffer with protease inhibitor PMSF (Beyotime). The lysates were resolved by SDS/PAGE, and proteins were transferred onto PVDF membranes. For WB, the following antibodies were used: the anti-actin (1:5000) was purchased from Abcam, anti-LC3B (1:2000) and anti-ULK1 (1:1000) were obtained from Sigma-Aldrich, anti-pULK1 (S555) (1:1000), and anti-pULK1 (S757) (1:1000) were purchased from Sigma, and anti-mTOR (1:1000), anti-mTOR (1:1000), anti-AMPK (1:1000), anti-p-AMPK (1:1000) were purchased from Cell Signaling Technology.

anti-ACC, (Milipore, #05-1098) anti-p-ACCSer79 (CST, #3661), anti-AMPKα (CST, #2793) anti-p-AMPKαThr172 (CST, #2535) anti-β-catenin (CST #8480), anti-CKB (Abclonal, #A12631), anti-CMKT2 (Abcam, #55963), anti-ERK (CST, #4696), anti-p-ERKThr202/Tyr204 (CST, #9101), anti-FAS (CST, #3180), anti-GAPDH (Abcam, #9485), anti-HSL (CST, #4107), anti-HSLSer563 (CST, #4139), anti-Perilipin1 (Abcam, #3526), anti-PPARγ (CST, #2435), anti-SERCA1 (Abcam, #2819), anti-SERCA2 (Invitrogen, #MA3-919), anti-SMAD2/3 (CST, #8685), anti-p-SMAD2Ser465/467 (CST, #3108), anti-p-SMAD3Ser423/425 (CST, #9520), anti-UCP1 (Abcam, #10983), anti-ULK1 (Sigma, #A7481), anti-p-ULK1Ser555 (CST #5869), anti-Vinculin (Sigma, #V9131).