Atg13

Manufactured by Cell Signaling Technology

Sourced in United States

ATG13 is a protein involved in the regulation of autophagy, a cellular process that degrades and recycles damaged or unnecessary cellular components. ATG13 is a key component of the ULK1 complex, which plays a crucial role in the initiation of autophagy. The core function of ATG13 is to facilitate the activity and regulation of the ULK1 complex, thereby contributing to the overall process of autophagy.

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Lab products found in correlation

18 protocols using atg13

Autophagy Regulatory Pathway Analysis

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Hydrogen peroxide, bafilomycin, antifade gold mounting medium, FBS,
L-glutamine, penicillin /streptomycin, DMEM without glucose, DMEM without
sodium pyruvate for H2O2 treatment medium, PVDF membrane, DMSO cell culture
grade, Lipofectamine 2000, Lipofectamine 3000 were purchased from Thermo
Fisher Scientific. Polyfect was purchased from qiagen. Antibodies for actin,
LC3, ATG5, myc, VPS34, ULK ser 317, ULK ser 757, ATG 13, ATG 13pSer 318,
Beclin1, Beclin Ser 15, VPS34 Ser 249, AMPK, PAMPK Thr 172,Flag, GST,
HA,FIP200, ATG101 were purchased from Cell Signaling Technology. ULK1ser555
and ULK ser 777 were from Millipore. ULK1 for immunoprecipitation was
purchased from Sigma. ULK1 for western blots was purchased from Santacruz
Biotechnology. Anti-mouse IPMK was developed in our lab.

Guha P., Tyagi R., Chowdhury S., Reilly L., Fu C., Xu R., Resnick A.C, & Snyder S.H. (2019). IPMK Mediates Activation of ULK Signaling and Transcriptional Regulation of Autophagy Linked to Liver Inflammation and Regeneration. Cell reports, 26(10), 2692-2703.e7.

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Comprehensive Western Blot Protocol

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Antibodies from Cell Signaling Technology are as follows: AMPKα (1:1000; #2532), p-AMPK^Thr172 (1:1000; #2531), ATG13 (1:1000; #13468), Crkl (1:500; #3182), p-Crkl^Tyr207 (1:100; #3181), mTOR (1:500; #2983), p-mTOR^Ser2448 (1:500; #2971), RPS6 (1:1000; #2317), p-RPS6^Ser240/244 (1:1000; #5364), ULK1 (1:1000; #8054), p-ULK1^Ser757 (1:500; #6888), p-ULK1^Ser555 (1:500; #5869), p-ATG13S^er318/ATG^Ser355 (1:1000; #46329), LC3B (1:500; #2775), ATG7 (1:1000; #8558), glyceraldehyde phosphate dehydrogenase (GAPDH) (1:1000; #5174), β-tubulin (1:1000; #2146), mouse immunoglobulin G (IgG) (1:5000; #7076), and rabbit IgG (1:5000; #7074). OXPHOS cocktail (1:2000; Abcam, #110413), green fluorescent protein (1:1000; Roche, #118144600001), MT-CO2 (1:2000; Thermo Fisher Scientific, #A-6404), p62 (1:1000; BD, #610832), and p-ATG13^Ser318 (1:1000; Abnova, #NBP2-19127) were also used.

Ianniciello A., Zarou M.M., Rattigan K.M., Scott M., Dawson A., Dunn K., Brabcova Z., Kalkman E.R., Nixon C., Michie A.M., Copland M., Vetrie D., Ambler M., Saxty B, & Helgason G.V. (2021). ULK1 inhibition promotes oxidative stress–induced differentiation and sensitizes leukemic stem cells to targeted therapy. Science translational medicine, 13(613), eabd5016.

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Western Blot and Immunoprecipitation Analysis

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Western blot and immunoprecipitation analysis were performed as described previously [13 (link)]. The antibodies for GAPDH (sc-47724; 1 : 1000), p62 (sc-101542; 1 : 1000), anti-LC3 (sc-398822; 1 : 1000), and individual secondary antibodies were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). The antibodies for ATG5 (#12994; 1 : 1000), ATG13 (#13468; 1 : 1000), ULK1 (#8054; 1 : 1000), and NEDD4L (#4013; 1 : 1000) were all obtained from Cell Signaling Technology (Cell Signaling Technology Inc., Beverly, USA).

Guo Y., Sun W., Gao W., Li L., Liang Y., Mei Z., Liu B, & Wang R. (2022). Long Noncoding RNA H19 Derived from M2 Tumor-Associated Macrophages Promotes Bladder Cell Autophagy via Stabilizing ULK1. Journal of Oncology, 2022, 3465459.

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Molecular Mechanism of Programmed Cell Death

Cited 2 times

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MMF was purchased from Selleckchem (Houston, TX). Neratinib was kindly supplied by Puma Biotechnology Inc. (Los Angeles, CA). Fingolimod (FTY720) was purchased from Sigma-Aldrich (St. Louis MO). Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Other reagents and performance of experimental procedures were as described (15 (link), 20 (link)–24 (link)). Antibodies used: AIF (5318), BAX (5023), BAK (12105), BAD (9239), BIM (2933), BAK1 (12105), Beclin1 (3495), cathepsin B (31718), CD95 (8023), FADD (2782), eIF2α (5324), P-eIF2α S51 (3398), ULK-1 (8054), P-ULK-1 S757 (14202), P-AMPK S51 (2535), AMPKα (2532), P-ATM S1981 (13050), ATM (2873), ATG5 (12994), mTOR (2983), P-mTOR S2448 (5536), P-mTOR S2481 (2974), ATG13 (13468), MCL-1 (94296), BCL-XL (2764), P-AKT T308 (13038), P-ERK1/2 (5726), P-STAT3 Y705 (9145), P-p65 S536 (3033), p62 (23214), LAMP2 (49067) all from Cell Signaling Technology; P-ULK-1 S317 (3803a) from Abgent; P-ATG13 S318 (19127) from Novus Biologicals.

Dent P., Booth L., Roberts J.L., Poklepovic A, & Hancock J.F. (2020). Fingolimod Augments Monomethylfumarate Killing of GBM Cells. Frontiers in Oncology, 10, 22.

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Antibody-Based Autophagy and Apoptosis Analysis

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The following antibodies were used: cleaved caspase-3 (Cell Signaling Technology), cleaved PARP (Cell Signaling Technology), LC3B (Cell Signaling Technology), β-actin (Sigma-Aldrich), FIP200 (ProteinTech Group), Atg13 (Cell Signaling Technology), BrdU (Developmental Studies Hybridoma Bank), goat anti-rabbit FITC (Jackson ImmunoResearch Laboratories). The following reagents were used: bafilomycin A1, spautin-1, paclitaxel, epirubicin, cisplatin, puromycin, BrdU, trichloroacetic acid, sulphorhodamine B, protease inhibitor cocktails, DAPI were from Sigma-Aldrich. MitoTracker Green FX (Invitrogen) and MitoTracker Red CMXRos (Invitrogen), Permount SP15-100 Toluene Solution (Fisher Scientific), DNase I (Roche).

Wen J., Yeo S., Wang C., Chen S., Sun S., Haas M.A., Tu W., Jin F, & Guan J.L. (2015). Autophagy inhibition re-sensitizes pulse stimulation-selected paclitaxel-resistant triple negative breast cancer cells to chemotherapy-induced apoptosis. Breast cancer research and treatment, 149(3), 619-629.

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Protein Extraction and Western Blotting

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One hundred microliters RIPA lysis buffer (Beyotime, Zhejiang, China) containing 1 μL PMSF (Sigma, USA) was used to extract the cellular proteins. Concentrations of proteins were measured by BCA kit (Thermo Scientific, USA). Antibodies against Livin (Cell Signaling Technology, USA), H2A.X (Abcam, USA), H2A.XY142F (Abcam, USA), GAPDH (Santa Cruz, USA), ATG5 (Cell Signaling Technology, USA), ATG13 (Cell Signaling Technology, USA), ATG14 (Cell Signaling Technology, USA), LC3-I/II (Abcam, USA) and β-actin (Santa Cruz, USA) were used at 1:1000 dilution.

Ge Y., Liu B.L., Cui J.P, & Li S.Q. (2019). Livin Regulates H2A.XY142 Phosphorylation and Promotes Autophagy in Colon Cancer Cells via a Novel Kinase Activity. Frontiers in Oncology, 9, 1233.

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Western Blot Analysis of Autophagy Proteins

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Western blotting was performed as described previously [18 (link)]. Primary antibodies [anti-GAPDH, 1:5000, Proteintech, China; anti-LC3-I, -LC3-II, -p-ULK1, -ATG13, -ULK1, 1:1000, Cell Signaling Technology, America] were used according to the manufacturer’s instructions. Image J software was employed to analyze relative protein expression.

Wang X., Lan Z., He J., Lai Q., Yao X., Li Q., Liu Y., Lai H., Gu C., Yan Q., Fang Y., Zhang Y., Li A, & Liu S. (2019). LncRNA SNHG6 promotes chemoresistance through ULK1-induced autophagy by sponging miR-26a-5p in colorectal cancer cells. Cancer Cell International, 19, 234.

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Fluorescent Probes for Autophagy and Mitophagy

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Mito-Tracker Green (#M7514, MTG) and Lyso-Tracker Red (#L12492, LTR) were obtained from Invitrogen (Eugene, Oregon, USA). Autophagosome dye (#NY561, DAPGreen) and mitophagy dye (#MD01-10, Kumamoto, Japan) were obtained from Dojindo Laboratories. Carbonyl cyanide 3-chlorophenylhydrazone (#045200, CCCP) was obtained from Thermo Fisherscientific (Grand Island, NY, USA). Penicillin-streptomycin (#15140163, 10,000 units/ml), fetal bovine serum (#26140079, FBS), Dulbecco’s modified Eagle’s medium (#11965118, DMEM), and other cell culture reagents were obtained from Gibco BRL (Grand Island, NY, USA). Primary and secondary antibodies used in this study were GAPDH (#5174, Cell Signaling Technology, Beverly, MA, USA), FIP200 (#12436, Cell Signaling Technology, Beverly, MA, USA), ATG13 (#13273, Cell Signaling Technology, Beverly, MA, USA), and HRP-linked anti-rabbit IgG (#7074, Cell Signaling Technology, Beverly, MA, USA). HeLa cells were a generous gift from Dr. Carolyn M. Price’s lab (University of Cincinnati). SLC-80 cells were isolated from healthy human fibroblasts by Dr. Taosheng Huang (Cincinnati Children’s Hospital, Cincinnati, OH, USA).

Chen Q., Shao X., Hao M., Fang H., Guan R., Tian Z., Li M., Wang C., Ji L., Chao H., Guan J.L, & Diao J. (2020). Quantitative analysis of interactive behavior of mitochondria and lysosomes using structured illumination microscopy. Biomaterials, 250, 120059.

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Skeletal Muscle Protein Analysis

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Skeletal muscle samples were prepared, processed, and analyzed as previously described by Lira et al. (36 (link)). The following antibodies and respective dilutions were used for immunoblots: sequestosome 1 (SQSTM1) (p62) (P0067, 1:1000), ULK1 (A7481, 1:1000), FLAG (F1804, 1:1000) from MilliporeSigma, ULK2 (HPA009027, 1:500) from MilliporeSigma, next to breast cancer type 1 susceptibility protein gene 1 protein (NBR1) (sc-130380, 1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA), LC3A/B (4108, 1:1000), ubiquitin (3936, 1:1000), cathepsin B (CTSB) (31718, 1:1000), lysosomal associated membrane protein 1 (LAMP1) (9091, 1:1000), autophagy related protein (Atg)13 (13273, 1:1000), Atg14 (5504, 1:1000), phosphorylated (p-)Atg14 (S29) (13155, 1:1000) from Cell Signaling Technology (Danvers, MA, USA), ULK2 (NBP1-33136, 1:500) from Novus Biologicals, p-p62 (S403) (MABC186-I, 1:1000) from MilliporeSigma, p-Atg13 (S318) (600-401-C49S, 1:1000) from Rockland (Limerick, PA, USA), and 20S (ab22673, 1:1000) from Abcam (Cambridge, United Kingdom). Results for protein expression were normalized to Ponceau stain.

Fuqua J.D., Mere C.P., Kronemberger A., Blomme J., Bae D., Turner K.D., Harris M.P., Scudese E., Edwards M., Ebert S.M., de Sousa L.G., Bodine S.C., Yang L., Adams C.M, & Lira V.A. (2019). ULK2 is essential for degradation of ubiquitinated protein aggregates and homeostasis in skeletal muscle. The FASEB Journal, 33(11), 11735-11745.

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Autophagy Regulation in Cell Lines

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Diphenyleneiodonium chloride (DPI), Dimethylsulfoxide (DMSO), and Earle's balanced salt solution (EBSS) were purchased from Sigma. Bafilomycin A1 was purchased from Santa cruz. The following rabbit antibodies were used: ULK1 (Cell Signaling), Atg13 (E1Y9V, Cell Signaling), Atg16L1 (Thermo Scientific, MBL), Beclin-1 (Santa Cruz), CD63 (Santa Cruz), LC3 (Sigma, MBL), ndp52 (Abcam). The following mouse antibodies were used: β-actin (Abcam, Santa Cruz), IgG1 K isotype (eBioscience), Galectin-3 (BD Pharmingen), p62 (BD Transduction Laboratories), TLR2 (Invivogen), ubiquitin (FK2, Enzo Life Sciences).

Bah A., Lacarrière C, & Vergne I. (2016). Autophagy-Related Proteins Target Ubiquitin-Free Mycobacterial Compartment to Promote Killing in Macrophages. Frontiers in Cellular and Infection Microbiology, 6, 53.

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