Atg9a

Manufactured by Cell Signaling Technology

Sourced in United States, Japan

ATG9A is a protein involved in the process of autophagy, which is the cellular mechanism for the degradation and recycling of damaged or unwanted cellular components. ATG9A is a transmembrane protein that plays a crucial role in the formation and trafficking of autophagosomes, the double-membrane vesicles that engulf the cargo for degradation. The core function of ATG9A is to facilitate the delivery of membrane components to the growing autophagosome.

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Lab products found in correlation

Caspase 3, by Cell Signaling Technology (2 mentions) Atg16l1, by Cell Signaling Technology (2 mentions) Fip200, by Cell Signaling Technology (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Gapdh, by Merck Group (1 mentions) Procaspase 3, by Cell Signaling Technology (1 mentions) Lamp2, by Santa Cruz Biotechnology (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Cl x posure film, by GE Healthcare (1 mentions) Amersham imager, by Cytiva (1 mentions) Amersham ecl, by Cytiva (1 mentions) Bca protein quantitative kit, by Beyotime (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Ripa buffer, by Beyotime (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Vegfa, by Abcam (1 mentions) Mmp 9, by Abcam (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Anti-ATG9A (2 citations)

7 protocols using atg9a

Quantitative Protein Analysis Protocol

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Total cell protein was isolated using RIPA buffer (Thermo Scientific, PI-89900) and quantified using BCA Protein Assay Kit (Pierce Biotechnology, 23225). Protein samples were run through SDS-PAGE and were transferred to polyvinylidene fluoride membrane, followed by a block in 10% dried non-fat milk, and then probed with primary antibodies against Caspase-3 (Novus Biological, 9662S), Procaspase-3 (Cell Signaling, 9662S), LC3B (Novus Biological, NB100-2220), SQSTM1/p62 (Thermo Scientific, H00008878-M01), LAMP-2 (Santa Cruz, sc-18822), ATG9A (ab108338), TFEB (Cell Signaling, 4240), GAPDH (Millipore, MAB374) and corresponding HRP conjugated goat anti-rabbit (Santa Cruz, sc-2004), goat anti-mouse (Bio-Rad, 172-1011), or donkey anti-goat (Santa Cruz, sc-2020) secondary antibodies. Proteins were visualized and quantified using chemiluminescent detection kit (GE Healthcare, Amersham ECL) and exposed to X-ray film (Thermo Scientific, CL-X Posure Film) or captured on an Amersham Imager 600 (GE Healthcare, CCD Model). The intensity for each band was densitometrically quantified and normalized against loading control using ImageJ software.

Kwon J.J., Willy J.A., Quirin K.A., Wek R.C., Korc M., Yin X.M, & Kota J. (2016). Novel role of miR-29a in pancreatic cancer autophagy and its therapeutic potential. Oncotarget, 7(44), 71635-71650.

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Protein Expression Analysis in Cancer Cells

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After transfection, 50–100 μl of RIPA buffer (Beyotime, Shanghai, China) was added to HeLa and SiHa cells to extract whole cell protein. The protein concentration was measured using a BCA protein quantitative kit (Beyotime). Following separation of 40 μg of protein by 8% SDS-PAGE, the protein was transferred onto a PVDF membrane (Millipore, Burlington, MA, USA) by wet transformation. Then, the PVDF membrane was incubated with 5% skimmed milk for 1 h. Next, specific antibodies against the target proteins were added and the membrane was incubated overnight at 4°C. The antibodies used were as follows: ATG9A, E-cadherin, Snail, Vimentin, caspase-3, LC3-II, P62, and Bax (Cell Signaling Technology, MA, USA); and VEGFA, MMP-2, MMP-9, and Bcl-2 (Abcam, MA, USA). Next, the membrane was incubated with secondary antibody labelled with horseradish peroxidase (Proteintech, Rosemont, IL, USA) for 1 h. Finally, the immunological complexes were developed by enhanced chemiluminescence (ECL, Hercules, CA, USA). At least three independent experiments were performed.

Liu X., Liu Z., Liu Y, & Wang N. (2023). ATG9A modulated by miR-195-5p can boost the malignant progression of cervical cancer cells. Epigenetics, 18(1), 2257538.

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Western Blot Analysis of ER Stress Pathway

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Equal amounts of protein (10-45 μg/lane) were loaded onto a 4-12% BIS-TRIS Bolt gel (Invitrogen) and run at 165V for 35 minutes. Protein was transferred to nitrocellulose membrane using the iBlot Western blotting system (Invitrogen). Immunodetection was performed using primary antibodies against STING, phosphorylated IRF3, TBK1, β-Actin (Abcam, Cambridge, MA), CRT, BIP/GRP78, PDI, caspase-3 and 12, ATF4, CHOP, BCL2, IRE1, XBP1, Atg9a, and acetylated lysine (Cell Signaling Technologies, Cambridge, MA) (all 1:1000 dilution) and the ECL Western Blotting Analysis System (Santa Cruz Biologicals, Santa Cruz, CA). Images were acquired using Multi-Gauge software (Fujifilm, Greenwood, SC).

Mitzel D.N., Lowry V., Shirali A.C., Liu Y, & Stout-Delgado H.W. (2014). Age Enhanced ER Stress Contributes to Increased Atg9A Inhibition of STING Mediated IFNβ Production during Streptococcus Pneumoniae Infection. Journal of immunology (Baltimore, Md. : 1950), 192(9), 4273-4283.

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Western Blot Analysis of Autophagy Regulators

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The cells were lysed in ice-cold whole cell extract buffer B (50 mM TRIS-HCl, pH 8.0, 4 M urea, and 1% Triton X-100) supplemented with protease inhibitor mixture. The cell extracts were resolved by SDS-PAGE and analyzed by western blotting. Protein bands were visualized using ECL Blotting Detection Reagents. All blots derive from the same experiment and they were processed in parallel. The antibodies used for western blotting include ATG3 (1:1000 dilution; 3415; Cell Signaling Technology), ATG5 (1:1000 dilution; 12994; Cell Signaling Technology), ATG7 (1:1000 dilution; 8558; Cell Signaling Technology), ATG9A (1:1000 dilution; 13509; Cell Signaling Technology), ATG16L1 (1:1000 dilution; 8089; Cell Signaling Technology), GCN5L2 (1:1000 dilution; 3305; Cell Signaling Technology), PCAF (1:1000 dilution; 3378; Cell Signaling Technology), P300 (1:1000 dilution; ab14984; Abcam), CBP (1:1000 dilution; ab253202; Abcam), SQSTM1 (1:1000 dilution; ab109012; Abcam), MAP1LC3A/B (1:1000 dilution; ab62721; Abcam), LAMP1 (1:1000 dilution; ab289548; Abcam), CTSB (1:1000 dilution; ab125067; Abcam), CTSD (1:1000 dilution; ab75811; Abcam), and β-Actin antibodies (1:1000 dilution; 60008-1-Ig; Proteintech).

Wang Z., Wang R., Niu L., Zhou X., Han J, & Li K. (2024). EPB41L4A-AS1 is required to maintain basal autophagy to modulates Aβ clearance. NPJ Aging, 10(1), 24.

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Comprehensive Antibody and Reagent Sources

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Antibodies used in this study were from the following sources: LC3B (#2775, rabbit, WB: 1:1,000), FIP200 (#12436, rabbit, WB: 1:1,000), ATG7 (#8558, rabbit, WB: 1:1,000), ATG9A (#13509, rabbit, WB: 1:1,000), ATG16L1 (#8089, rabbit, WB: 1:1,000), and Gal3 (#87985, rabbit, IF: 1:400) were purchased from Cell Signaling Technology. Anti-beta-actin antibody (A2228, mouse, WB: 1:10,000) was purchased from Sigma-Aldrich. LC3 (PM036, rabbit, IF: 1:500) antibody was purchased from MBL International. ALIX antibody (634502, mouse, IF: 1:200) was purchased from BioLegend. IST1 (51002-1-AP, rabbit, IF: 1:100) and CHMP2A (10477-1-AP, rabbit, IF: 1:199) antibodies were purchased from proteintech. Gal8 antibody (NBP2-75501, rabbit, IF: 1:400) was purchased from Novus Biologicals. Goat anti-rabbit-HRP (Cat# 31460, WB: 1:10,000) and goat anti-mouse-HRP (Cat# 31430, WB: 1:10,000) antibodies were purchased from Thermo Fisher Scientific. Alexa Fluor 488/568/647-conjugated secondary antibodies for immunofluorescence were purchased from Thermo Fisher Scientific.
Reagents used in this study were from the following sources: Filipin (F9765) from Sigma-Aldrich; Bafilomycin A1 (BML-CM110-0100) from Enzo.

Jia X., Knyazeva A., Zhang Y., Castro-Gonzalez S., Nakamura S., Carlson L.A., Yoshimori T., Corkery D.P, & Wu Y.W. (2022). V. cholerae MakA is a cholesterol-binding pore-forming toxin that induces non-canonical autophagy. The Journal of Cell Biology, 221(12), e202206040.

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Western Blot Analysis of Autophagy Proteins

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Cells were lysed in buffer (10 mM HEPES pH 7.4, 50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na₃VO₄, 0.1% Triton X-100) and Western blotting was conducted using the following primary antibodies: LC3B (Novus Biologicals, NB100-2220), β-actin (ACTB, Sigma-Aldrich, A5316), ATG5 (Santa Cruz, sc-33210), FIP200 (Cell Signaling Technology, D10D11), BECN1 (Santa Cruz, sc-48341), PIK3C3 (Santa Cruz, sc365404), WIPI2 (Cell Signaling Technology, #8567), ATG13 (Cell Signaling Technology, D4P1K), ATG9A (Cell Signaling Technology, D4O9D), ULK1 (Santa Cruz, sc-390904), ULK2 (GeneTex, GTX111476), UVRAG (Santa Cruz, sc-293268), ATG14 (Cell Signaling Technology, #5504), ATG16L1 (Santa Cruz, sc-393274), cleaved-caspase 3 (Cell Signaling Technology, #9661), viral capsid protein 1 (VP1, Dako, M706401-1), FLAG (Sigma, F1804), PI4KIIIβ (Sigma 06578), and PIKfyve (Santa Cruz, sc-100408). Blots were cropped to improve clarity and conciseness. Uncropped blots are provided in Supplemental Figure 2.

Mohamud Y., Shi J., Tang H., Xiang P., Xue Y.C., Liu H., Ng C.S, & Luo H. (2020). Coxsackievirus infection induces a non-canonical autophagy independent of the ULK and PI3K complexes. Scientific Reports, 10, 19068.

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Autophagy and Apoptosis Pathway Proteins

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The antibodies against autophagy-related proteins (Beclin1, ATG3, ATG 5, ATG 7, ATG 9A, ATG 12, ATG 13, LC3, and HSPB8), apoptosis-related proteins (caspase-9, cleaved caspase-9, caspase-7, cleaved caspase-7, caspase-3, and cleaved caspase-3) and RAB7 were purchased from Cell Signaling Technology (CST, Japan). The anti-actin antibody was purchased from Santa Cruz. The Alexa-conjugated anti-mouse and anti-rabbit IgG were purchased from Life Technologies. Small interfering RNA (siRNA) for ATG9A, HSPB8, LC3, RAB7 and non-targeting control siRNA were purchased from Thermo Scientific (Lafayette, CO). siRNAs for Beclin1 and ATG5 were purchased from CST. Gefitinib was purchased from Cayman chemical (MI).

Sato Y., Kubo T., Morimoto K., Yanagihara K, & Seyama T. (2016). High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells. BMC Cancer, 16, 63.

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