Anti atg14

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-ATG14 is a primary antibody that recognizes the ATG14 protein. ATG14 is a critical component of the autophagy-specific phosphatidylinositol 3-kinase (PI3K) complex and plays a key role in the initiation of autophagosome formation.

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7 protocols using anti atg14

Antibodies for Western Blot Analysis

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All primary antibodies used for western blots were purchased from commercial sources. Antibodies from Santa Cruz include: anti-beclin 1 (sc11427), anti-myc HRP (sc40 HRP) and anti-β-actin HRP (sc47778 HRP). Antibodies from Cell Signaling Technology include: anti-UVRAG (#11315), anti-Vps34 (#4263), anti-Atg14 (#96752, to detect mouse Atg14), anti-p-AMPKα(Thr172) (#2535), anti-AMPKα (#2532), anti-p-Raptor(Ser792) (#2083), anti-Raptor (#2280), anti-TBC1D1 (#66433) and anti-HA (#3724, to detect Tlr9-HA in tissue lysates of Tlr9-HA KI mice). Other primary antibodies include: anti-p-ACC(Ser79) (#07–303, Millipore), anti-ACC (#04–322, Millipore), anti-p-TBC1D1(Ser237) (#07–2268, Millipore), anti-Atg14 (M184–3, MBL International, to detect human ATG14), anti-HA high affinity-HRP (#12013819001, Roche, to detect overexpressed TLR9-HA in cells and Tlr9-HA after immunoprecipitation from muscle lysates of the Tlr9-HA KI mice), anti-Flag M2-HRP (A8592, Sigma), and anti-GLUT4 (GT41-A, Alpha Diagnonstic International). To detect beclin 1 in Tlr9-HA immunoprecipitates from muscle lysates by western blot analysis, 10% MINI-PROTEAN TGX Precast gels (Bio-Rad) were used. For detection of all other proteins, 4–20% Precast gels were used.

Liu Y., Nguyen P.T., Wang X., Zhao Y., Meacham C.E., Zou Z., Bordieanu B., Johanns M., Vertommen D., Wijshake T., May H., Xiao G., Shoji-Kawata S., Rider M.H., Morrison S.J., Mishra P, & Levine B. (2020). Tlr9 and Beclin 1 Cross-Talk Regulates Muscle AMPK Activation in Exercise. Nature, 578(7796), 605-609.

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Antibody Targeting of Lon Protease Autophagy Pathway

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Antibodies to human Lon were produced as described previously [35 (link)]. The following primary antibodies were used in this study: Autophagy Induction Antibody Sampler Kit containing Anti-ULK1 monoclonal antibody, anti-p-ULK1 S555, anti-ATG13, anti-p-ATG13 S355, anti-FIP200, anti-ATG101. Anti-NFkB anti-LC3B, anti-LAMP1, anti-VDAC, anti-Calnexin, anti-Tubulin, and ULK1 antibody sampler Kit containing anti-ATG14, anti-p-ATG14 S29, anti-Beclin1, anti-p-Beclin1 S15, were purchased from Cell signaling technology. Anti-FACL4, anti-GAPDH, and anti-β-actin were purchased from Gentex also the Gentex helped in producing in-house antibody anti-FUNDC1 and anti-p-FUNDC1 S17 which were raised in rabbit and purified. Anti-HSP60 and anti-Aconitase2 from santa cruz. Anti-myc and and anti-Flag were obtained from Merck Millipore. Anti-HIF1α was obtained from Life science bio. Anti-ULK1 for immuofluorescence studies was obtained from Santa cruz. Cobalt chloride (CoCl₂), SBI-0206965, Bafilomycin A1 and N-Acetyl Cysteine (NAC) were purchased from sigma, dissolved in DMSO and stored at −20 °C.

Ponneri Babuharisankar A., Kuo C.L., Chou H.Y., Tangeda V., Fan C.C., Chen C.H., Kao Y.H, & Lee A.Y. (2023). Mitochondrial Lon-induced mitophagy benefits hypoxic resistance via Ca2+-dependent FUNDC1 phosphorylation at the ER-mitochondria interface. Cell Death & Disease, 14(3), 199.

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Antibodies for Western Blot Analysis

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Rapamycin Modulates Autophagy Markers

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The reagent rapamycin (Rapa; R0395; Sigma-Aldrich) was used in the study. The primary antibodies used were anti-ATG14 (no. 96752; Cell Signaling Technology), anti-UVRAG (no. 13115; Cell Signaling Technology), anti-BECN1 (no. 54101; Cell Signaling Technology), anti-p62 (P0067; Sigma-Aldrich), anti-LC3 (L8918; Sigma-Aldrich), anti-Bif1 (NBP2-24733; Novus), anti-GAPDH (RM2002; Beijing Ray Antibody Biotech), anti-RABV (5B12) (NB110-7542; Novus), and fluorescein isothiocyanate (FITC)-conjugated anti-RABV (800-092; FUJIREBIO). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (ab97051; Abcam) and goat antimouse IgG (sc-2005; Santa Cruz).

Hou P., Guo Y., Jin H., Sun J., Bai Y., Li W., Li L., Cao Z., Wu F., Zhang H., Li Y., Yang S., Xia X., Huang P, & Wang H. (2023). Bif-1c Attenuates Viral Proliferation by Regulating Autophagic Flux Blockade Induced by the Rabies Virus CVS-11 Strain in N2a Cells. Microbiology Spectrum, 11(3), e03079-22.

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Western Blotting for Autophagy Proteins

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Western blotting was carried out as previously described,^{42 (link)} using anti-LC3 (Abcam, Cambridge, UK), anti-ATG2A (Cell Signaling Technology Inc., Danvers, MA, USA), anti-ATG14 (Cell Signaling Technology Inc.), and anti-YTHDF1 (Proteintech, Wuhan, China). Secondary antibodies were obtained from Sigma-Aldrich.

Li Q., Ni Y., Zhang L., Jiang R., Xu J., Yang H., Hu Y., Qiu J., Pu L., Tang J, & Wang X. (2021). HIF-1α-induced expression of m6A reader YTHDF1 drives hypoxia-induced autophagy and malignancy of hepatocellular carcinoma by promoting ATG2A and ATG14 translation. Signal Transduction and Targeted Therapy, 6, 76.

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Autophagy Regulatory Protein Antibody Protocol

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Antibodies used in this study included anti-ATG14 (#5504, Cell Signaling), anti-Flag M2 (F3165, Sigma), anti -HA (12CA5, Roche), anti-EGFP (GL-8, Clontech), anti-LC3 (7543, Sigma), anti-p62 (PM045, MBL), anti-ATG16 (PM040, M150-3, MBL), anti-STX17 (HPA001204, Sigma), anti-beclin 1 (sc-11427, Santa Cruz), anti-Tom20 (Ab78547, Abcam), anti-LAMP2 (sc-18822, Santa Cruz), anti-Myc (9E10, DSHB) and anti-β-tubulin (E7, DSHB). U₂OS and HEK293T were described before^{25 (link)}.

Diao J., Liu R., Rong Y., Zhao M., Zhang J., Lai Y., Zhou Q., Wilz L.M., Li J., Vivona S., Pfuetzner R.A., Brunger A.T, & Zhong Q. (2015). ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature, 520(7548), 563-566.

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Autophagy Pathway Protein Analysis

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Anti-Atg14 (5504S, Cell Signaling, Danvers, MA, USA), Anti-LAMP2 (PRS3627, Sigma-Aldrich, St. Louis, MO, USA), Anti-LAMP2 (ab13524, Abcam, Cambridge, UK), Anti-LC3 (SAB1305552, Sigma-Aldrich), Anti-LC3 (66139-1-lg, Proteintech, Rosemont, IL, USA), Anti-mTOR (#2972, Cell Signaling), Anti-Phospho-mTOR (Ser2481) (#2974S, Cell Signaling), Anti-Phospho-mTOR (Ser2448) (#5536, Cell Signaling), Anti-p62 (ab155686, Abcam), Anti-STX17 (HPA001204, Sigma-Aldrich).

Hua R., Han S., Zhang N., Dai Q., Liu T, & Li J. (2018). cPKCγ-Modulated Sequential Reactivation of mTOR Inhibited Autophagic Flux in Neurons Exposed to Oxygen Glucose Deprivation/Reperfusion. International Journal of Molecular Sciences, 19(5), 1380.

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