Rabbit anti atg7

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Rabbit anti-ATG7 is a primary antibody that specifically recognizes the ATG7 protein. ATG7 is an essential component of the autophagy pathway and plays a critical role in the formation of autophagosomes. This antibody can be used to detect and quantify the expression of ATG7 in various cellular and tissue samples.

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Anti-ATG7 (85 citations)

14 protocols using rabbit anti atg7

Quantitative Immunoblotting for Cellular Signaling

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Samples were lysed in RIPA lysis buffer (Thermo Scientific) supplemented with a protease inhibitor (Roche). Protein concentrations were measured using the Bradford Assay (Bio-Rad). Equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked in 5% nonfat milk and incubated overnight at 4°C in blocking buffer containing primary antibodies. Antibodies used for immunoblotting were: rabbit anti-LC3B (1:1000; Sigma), rabbit anti-p21 (1:1000; Cell Signaling), rabbit anti-p27 (1:1000; Cell Signaling), rabbit anti-cyclin D1 (1:1000; Cell Signaling), mouse anti-p62 (1:1000; Abcam), rabbit anti-ERα (1:1000; Cell Signaling), rabbit anti-Atg7 (1:1000; Cell Signaling), rabbit anti-Beclin1 (1:1000; Cell Signaling), rabbit anti-ILK1 (1:100; Cell Signaling), and rabbit anti-GAPDH (1:2000; Cell Signaling). After three washes with TBST, blots were probed with horseradish peroxidase-conjugated anti-rabbit secondary antibodies for 45 min (1:5000; Cell Signaling). Following incubation with ECL Plus Western Blotting Detection System (GE Healthcare), blots were imaged using a FluorChemE Imager (Cell Biosciences). For each protein, densitometry analysis was carried out in Image J by dividing the total intensity of each band by that of GAPDH in the same sample.

Anlaş A.A, & Nelson C.M. (2020). Soft microenvironments induce chemoresistance by increasing autophagy downstream of integrin-linked kinase. Cancer research, 80(19), 4103-4113.

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Comprehensive Antibody Panel for Autophagy and UPR Analysis

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Rabbit anti-LC3B (#3868), rabbit anti-ATG5 (#12994), rabbit anti-ATG7 (#8558), rabbit anti-Beclin-1 (#3495), rabbit anti-ATG16L1 (#8089), rabbit anti-BIP (#3177), rabbit anti-ATF6 (#65880), rabbit anti-ATF4(#11815), rabbit anti-XBP1 (#12782), rabbit anti-PERK (#5683), rabbit anti-IRE1 (#3294), rabbit anti-p-eif2α (#3398), rabbit anti-FIP200 (#12436), and mouse anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (dilution concentration 1:1000). Rabbit anti-p-IRE1 (PA1-16927) was purchased from Invitrogen (Waltham, MA, USA) (dilution concentration 1:1000). Mouse anti-p62 (18420-1-AP), mouse anti-HA-tag (66006-2-Ig), and mouse anti-β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China) (dilution concentration 1:5000). HRP-conjugated goat anti-mouse (G1214) and goat anti-rabbit (G1213) were purchased from Servicebio (Wuhan City, China) (dilution concentration 2:5000). Alexa Fluor 568 goat anti-mouse IgG, IgM (H + L) (A-11004) and Alexa Fluor 647 goat anti-mouse-IgG (H + L) (A-21445) were purchased from Thermo Fisher (Waltham, MA, USA) (dilution concentration 1:500). Immunofluorescence antibodies were diluted in PBS. Immunoblot antibodies were diluted in TBST.

Su W.Q., Yu X.J, & Zhou C.M. (2021). SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response. Viruses, 13(12), 2467.

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Antibody and Chemical Reagents for PRRSV Study

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Primary antibodies were mouse anti-PRRSV N protein (produced in our laboratory), mouse anti-β-actin (Proteintech, #A5316), mouse anti-Flag (Proteintech, #80010-1-RR), rabbit anti-LC3 (Proteintech, #14600-1-AP), rabbit anti-ATG7 (Cell Signaling Technology, #2631), rabbit anti-CaMKII (Abcam, #ab168818), rabbit anti-p-AMPK (Thr172) (Cell Signaling Technology, #2535), rabbit anti-AMPK (Cell Signaling Technology, #2532), rabbit anti-p-mTOR(Ser2448) (Cell Signaling Technology, #5536), rabbit anti-mTOR (Cell Signaling Technology, #2983), rabbit anti-STIM1 (Cell Signaling Technology, #4916), mouse anti-Orai1 (Proteintech, #66223–1), rabbit anti-GRP78 (Proteintech, #11587-1-AP) and rabbit anti-Calnexin (Abcam, #ab92573). HRP-labeled rabbit or mouse secondary antibodies were purchased from Beyotime (China).
Chemicals used included 2-Aminoethyl diphenylborinate (2-APB) (Selleck, #S6657), 3-MA (Selleck, #S2767), 4-Phenylbutyric acid (4-PBA) (Selleck, #S3592), BAPTA-AM (Selleck, #S7534), Dantrolene sodium (Selleck, #S5478), Dorsomorphin (Compound C) 2HCL (Selleck, #S7306), KN-93 (Selleck, #S7423), ML-9 HCL (Selleck, #S6847), Procaine (Selleck, #S4668), Rapamycin (MedChemExpress, #HY-10219), Tetracaine-HCL (Selleck, #S2573), Thapsigargin (Selleck, #S7895), Tunicamycin (Beyotime, #SC0393).

Diao F., Jiang C., Sun Y., Gao Y., Bai J., Nauwynck H., Wang X., Yang Y., Jiang P, & Liu X. (2023). Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication. PLOS Pathogens, 19(3), e1011295.

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Western Blot Analysis of Autophagy and Apoptosis Markers

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Total protein was extracted from cells using RIPA buffer (Biosharp, China), and then the protein concentration was quantified using a bicinchoninic (BCA) protein assay kit (Beyotime, China). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat milk for 1 h and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit anti-LC3 (Cell Signaling Technology, 12741), rabbit anti-Beclin1 (Cell Signaling Technology, 3495), rabbit anti-ATG7 (Cell Signaling Technology, 8558), rabbit anti-P662 (Cell Signaling Technology, 16177), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, 9664), rabbit anti-caspase 3 (Cell Signaling Technology, 14220), rabbit anti-Bax (BOSTER, A00183), rabbit anti-Bcl-2 (BOSTER, A00040-2), rabbit anti-β-actin (BOSTER, BA2305). Afterward, the membranes were incubated with secondary antibody at room temperature for 1 h. An ECL kit (Beyotime Biotechnology, Shanghai, China) was used to detect the bands of the western blots.

Qiu Z., Wang Y., Liu W., Li C., Zhao R., Long X., Rong J., Deng W., Shen C., Yuan J., Chen W, & Shi B. (2021). CircHIPK3 regulates the autophagy and apoptosis of hypoxia/reoxygenation-stimulated cardiomyocytes via the miR-20b-5p/ATG7 axis. Cell Death Discovery, 7, 64.

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Autophagy Marker Immunoblotting Protocol

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The specific method is as mentioned before [28 (link)]. In this study, primary antibodies included rabbit anti-LC3B (1:1000; Sigma-Aldrich L7543), rabbit anti-P62 (1:1000; Abcam ab205719), mouse anti-β-Actin (1:1000; Sigma-Aldrich A5316), rabbit anti-Sidt2 (1:1000; Invitrogen PA5-69064), rabbit anti-Atg5 (1:1000; Cell Signaling Technology 9980S), rabbit anti-Atg7 (1:1000; Cell Signaling Technology 8558S), rabbit anti-Atg12 (1:1000; Cell Signaling Technology 2011S), mouse anti-LAMP1 (1:1000; Santa Cruz Biotechnology H4A3), rabbit anti-cathepsin B (1:1000; Cell Signaling Technology 31718S).

Geng M.Y., Wang L., Song Y.Y., Gu J., Hu X., Yuan C., Yang M., Pei W.J., Zhang Y, & Gao J.L. (2021). Sidt2 is a key protein in the autophagy-lysosomal degradation pathway and is essential for the maintenance of kidney structure and filtration function. Cell Death & Disease, 13(1), 7.

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Autophagy Regulation in Rat Mast Cells

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The HBZY-1 rat MC line was obtained from the Chinese Center for Type Culture Collection (Wuhan, China). Chloroquine (CQ), 3-methyladenine (3-MA) and Aldo were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Mouse anti-β-actin (catalog no. 3700), rabbit anti-Atg5 (catalog no. 12994), rabbit anti-Atg7 (catalog no. 8558), rabbit anti-LC3 (catalog no. 3868) and rabbit anti-p62 (catalog no. 5114) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). All other chemicals were of analytical grade.

Yang M., Wang B., Miao L., Xu X, & He X. (2016). Autophagy is involved in aldosterone-induced mesangial cell proliferation. Molecular Medicine Reports, 14(5), 4638-4642.

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Antibody Characterization for Autophagy Study

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The following antibodies were used in the present study: rabbit anti-ATG9A,²⁸ (link) guinea pig anti-SQSTM1 (C-terminal; Progen, GP62-C), rabbit anti-SQSTM1 (MBL, PM045), mouse anti-NBR1 (Abcam, ab55474), rabbit anti-LC3A/B (Cell Signaling Technology, 4108), mouse anti-GOLGA2/GM130 (BD Biosciences, 610822), mouse anti-ACTB/ß-actin (Sigma-Aldrich, A5441), rabbit anti-ubiquitin (Dako, Z0450), mouse anti-ubiquitin (P4D1; Santa Cruz Biotechnology, sc-8017), mouse anti-GFAP (Sigma-Aldrich, G3893), rabbit anti-AIF1/Iba1 (Wako Pure Chemical Industries, 019–19741), goat anti-VGAT (Frontier Institute, VGAT-Go-Af620), goat anti-CALB/calbindin (Frontier Institute, Calbindin-Go-Af1040), rat anti-myelin basic protein (AbD Serotec, MCA409), mouse anti-NEFH/Neurofilament heavy (clone SMI31; BioLegend, 80163), rabbit anti-ATG7 (Cell Signaling Technology, 8558), and goat anti-GFP (Frontier Institute, GFP-Go-Af1480–1).

Yamaguchi J., Suzuki C., Nanao T., Kakuta S., Ozawa K., Tanida I., Saitoh T., Sunabori T., Komatsu M., Tanaka K., Aoki S., Sakimura K, & Uchiyama Y. (2018). Atg9a deficiency causes axon-specific lesions including neuronal circuit dysgenesis. Autophagy, 14(5), 764-777.

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Immunoblotting Techniques for Cellular Signaling

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All media (RPMI, DMEM) and PBS buffers were purchased from Gibco (61870–010; 41966–029; 14190–094). NaCl was purchased from Merck. LPS (E. coli O111:B4) and DPI were purchased from Sigma Aldrich (D2926). Torin1 was purchased from Tocris (4247); E-64d was obtained from Hycultec (HY-15282). Ionomycin (407952) was purchased from CalbioChem. ATP (tlrl-atpl) and bafilomycin A₁ (tlrl-baf1) were obtained from Invivogen. The prolyl-hydroxylase inhibitor DMOG was purchased from Cayman Chemical (71210). For immunoblotting, the following antibodies were used: rabbit anti-ACT (A2066; Sigma-Aldrich); rabbit-anti-HSP90A/B (Santa Cruz Biotechnology, sc-7947), rabbit anti-NFAT5 (Thermo Scientific, PA1-023), rabbit anti-MAP1LC3B (Novus Biologicals, NB100-2220), rabbit anti-ATG7 (Cell Signaling Technology, D12B11) rabbit anti-p-AKT (Cell Signaling Technology, 4060S), rabbit anti-AKT (Cell Signaling Technology, 9272S), rabbit anti-SQSTM1 (Sigma Aldrich, P0067), rabbit anti-HIF1A (Cayman Chemical, 10006421). As secondary antibody, we used swine anti-rabbit HRP (P0399, Dako).

Neubert P., Weichselbaum A., Reitinger C., Schatz V., Schröder A., Ferdinand J.R., Simon M., Bär A.L., Brochhausen C., Gerlach R.G., Tomiuk S., Hammer K., Wagner S., van Zandbergen G., Binger K.J., Müller D.N., Kitada K., Clatworthy M.R., Kurts C., Titze J., Abdullah Z, & Jantsch J. (2019). HIF1A and NFAT5 coordinate Na+-boosted antibacterial defense via enhanced autophagy and autolysosomal targeting. Autophagy, 15(11), 1899-1916.

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Western Blot Analysis of Autophagy Markers

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The proteins were harvested and lysed in RIPA buffer. Equal amounts of protein extracts (30 μg) were loaded per lane and resolved by SDS/PAGE. Subsequently, polypeptides were separated and transferred to PVDF membranes. The membranes were blocked and then treated overnight with rabbit anti-Beclin-1 (1 : 1000, Cell Signaling Technology), rabbit anti-Atg7 (1 : 1000, Cell Signaling Technology), rabbit anti-Atg5 (1 : 1000, Cell Signaling Technology), rabbit anti-P62 (1 : 1000, Cell Signaling Technology), rabbit anti-LC3II/I (1 : 1000, Sigma), and mouse anti-β-actin (1 : 1000, Beyotime Institute of Biochemistry) antibodies, respectively. After being washed with TBST three times, the membranes were incubated using the corresponding HRP-conjugated secondary antibodies (1 : 1000, Beyotime Institute of Biochemistry) with blocking solution at room temperature for 2 h. Finally, the expression levels of protein were measured by enhanced chemiluminescence kit (Millipore). The protein bands were normalized by β-actin and quantified as the ratio of the optical density.

Liu X., Gao C., Wang Y., Niu L., Jiang S, & Pan S. (2021). BMSC-Derived Exosomes Ameliorate LPS-Induced Acute Lung Injury by miR-384-5p-Controlled Alveolar Macrophage Autophagy. Oxidative Medicine and Cellular Longevity, 2021, 9973457.

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Western Blot Analysis of Autophagy Markers

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Western blotting was performed as previously described29 (link). Equal amounts of protein were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight with primary rabbit anti-LC3B (1:1000, Abcam, Cambridge, UK), rabbit anti-Atg7 (1:500, Cell Signaling Technology), rabbit anti-HO-1 (1:1000, Abcam), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20000; Sigma-Aldrich). Blots were developed using a chemiluminescent substrate and visualized on the Kodak Image Station (Carestream Health Inc, Rochester, NY). Densitometry of bands in western blot was analyzed by Image J program (NIH, Bethesda, USA). The relative amount of each protein was normalized to the ratio of GAPDH.

Liu A., Huang L., Guo E., Li R., Yang J., Li A., Yang Y., Liu S., Hu J., Jiang X., Dirsch O., Dahmen U, & Sun J. (2016). Baicalein pretreatment reduces liver ischemia/reperfusion injury via induction of autophagy in rats. Scientific Reports, 6, 25042.

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