TBT and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell lysis buffer, fetal bovine serum (FBS), RPMI 1640 medium, and Western Bright Enhanced Chemiluminescence (ECL) detection reagents were obtained from Thermo Fisher Scientific (Suwanee, GA, USA). An Annexin V/PI apoptosis detection kit was purchased from Multi-Sciences (Hangzhou, China). Anti-Bip (Cat#3177), anti-Calnexin (Cat#2679), anti-Ero1-Lα (Cat#3264), anti-IRE1α (Cat#3294), anti-PDI (Cat#3501), anti-CHOP (Cat#2895), anti-PERK (Cat#5683), anti-eIF2α (Cat#5324), anti-P-eIF2α (Cat#3398), anti-Atg12 (Cat#4180), anti-Beclin-1 (Cat#3495), anti-JNK (Cat#3708), anti-P-JNK (Cat#4668), anti-LC3A/B (Cat#12741), anti-Atg5 (Cat#12994), anti-Atg16L1 (Cat#8089), anti-Atg7 (Cat#8558), anti-Atg3 (Cat#3415), anti-rabbit IgG (Cat#7074), and anti-mouse IgG (Cat#7076) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-ATF6 (Cat#ab227830) was purchased from Abcam (Cambridge, UK) and anti-GAPDH was obtained from KangChen Biotech (Shanghai, China).
Anti atg3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-ATG3 is a primary antibody that specifically binds to the ATG3 protein. ATG3 is an essential component of the autophagy pathway and is involved in the conjugation of Atg8/LC3 to phosphatidylethanolamine, a critical step in autophagosome formation.
Automatically generated - may contain errors
Lab products found in correlation
Anti atg7, by Cell Signaling Technology (8 mentions)
Anti atg5, by Cell Signaling Technology (7 mentions)
Anti beclin 1, by Cell Signaling Technology (5 mentions)
Anti atg12, by Cell Signaling Technology (5 mentions)
Anti lc3a b, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti atg16l1, by Cell Signaling Technology (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti chop, by Cell Signaling Technology (1 mentions)
Anti bip, by Cell Signaling Technology (1 mentions)
Anti ire1α, by Cell Signaling Technology (1 mentions)
Anti atf6, by Abcam (1 mentions)
Anti calnexin, by Cell Signaling Technology (1 mentions)
Anti pdi, by Cell Signaling Technology (1 mentions)
11 protocols using anti atg3
1
ER Stress-Induced Apoptosis and Autophagy
2
Transgenic Mice for Autophagy Analysis
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B6D2-Tg (CAG/Su9-DsRed2, Acr3-EGFP) RBGS002Osb (RBRC03743) (Hasuwa et al., 2010 (link)), MAP1LC3B-GFP (RBRC00806) (Mizushima et al., 2004 (link)), and Atg3 heterozygous (RBRC02761) (Sou et al., 2008 (link)) mice were bought from Riken BioResource Center. Mito-Tracker Red (40743ES50) was bought from Yeasen. The anti-MAP1LC3B antibody (Medical and Biological Laboratories Co., PM036) was used at 1:200; Alexa Fluor®488 donkey anti-rabbit IgG (H + L) (Invitrogen Thermo Fisher Scientific, A21206) was used at 1:500; anti-Actin (1:5,000) was obtained from Sigma Aldrich (A5441); anti-ATG3 (1:500) was purchased from Cell Signaling Technology (3415S); anti-PINK1 polyclonal antibody (NOVUS, BC100–494) was used at 1:1,000; Anti-OPTN polyclonal antibody (1:1,000) was bought from Proteintech Group (10837-1-AP); FCCP (C2920) and EBSS (E2888) were obtained from Sigma Aldrich. Rapamycin (HY-10219) and Brefeldin A (HY-16592) were bought from Med Chem Express. Mito-Keima vector was shared by Prof. Chen Quan (Wu et al., 2020 (link)). Tim23 antibody (BD Biosciences, 611223) was shared by Prof. Liu Lei.
3
Western Blot Analysis of Cellular Proteins
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Protein from the treated cell cultures was extracted and Western blot analysis was performed as described previously [37 (link)]. Expression of EBV lytic proteins was detected with anti-Zta (1:200; gift from Prof. P. Farrell, Imperial College, London, UK) and anti-BMRF1 (1:1000; gift from Dr.KH Chan, Department of Microbiology, HKU, Hong Kong SAR, China). Expression of phosphorylated ERK1/2 was detected with p-ERK1/2 rabbit polyclonal antibodies, (1:1000; #9101, Cell Signaling Technology Danvers, MA, USA). Expressions of ER stress markers PERK. IRE-1α, BiP, CHOP, XBP-1s was detected with ER stress sampler kit (1:1000; #9956, Cell Signaling Technology). Expression of autophagic proteins was detected with anti-ATG5 (1:1000; #12994, Cell Signaling Technology), anti-ATG7 (1:1000; #8558, Cell Signaling Technology,), anti-beclin-1 (1:1000; #3738, Cell Signaling Technology), anti-LC3B (1:1000; #2775, Cell Signaling Technology), anti-ATG10 (1:1000; #PA5-78593, Invitrogen), anti-ATG3 (1:1000; #3415, Cell Signaling Technology,), anti-ATG12 (1:1000; #4180, Cell Signaling Technology,) and anti-rab9 (1:1000; #5133. Cell Signaling Technology). Expression of human cellular α-tubulin and β-actin was detected with anti-α-tubulin and anti-β-actin antibody (1:5000; MilliporeSigma, Burlington, MA, USA), respectively, as loading controls.
4
Western Blot Analysis of Autophagy and DNA Repair Markers
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Cells were lysed in RIPA buffer and cleared by centrifugation (14,000 RPM for 15 min at 4°C). Protein concentration was determined using the bicinchoninic acid assay (BCA) method. Cell lysates containing 20 µg protein were separated by SDS-PAGE gel electrophoresis and transferred onto a Polyvinylidene difluoride membrane (BioRad). The membranes were blocked with 5 % skimmed milk powder (Sigma Aldrich) for 1 h at room temperature, washed with TBST, and reacted with primary antibody anti-LC3(A/B)-I/II, anti-Beclin, anti-ATG3, anti-ATG5, anti-ATG16, anti-H2AX, anti-SIRT1, anti-MLH1, anti-Lamin A/C, anti-PARP, or anti-GAPDH (all antibodies were from Cell Signaling Technology, MA, USA) at 1:1000 dilution overnight at 4°C. The specific HRP-labeled secondary antibodies (Cell Signaling Technology) were then reacted at 1:2000 dilutions for 1 h at room temperature. Chemiluminescence was detected using Enhanced Chemiluminescence western blotting detection reagent (BioRad, CA, USA). GAPDH or PARP was used as a loading control.
5
Quantifying Autophagy Proteins by Western Blot
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Protein quantities were analyzed by standard Western blotting technique. Briefly, 25-50 μg total proteins lysates were separated on Mini PROTEAN Precast Gels with 2 X Laemmli sample buffer (with 2.5% β-mercaptoethanol) to a final 30 μL volume. The proteins were transferred onto a PVDF-membrane and blocked with superblock solution and probed with anti-BECLIN1 (1:1000), anti-ATG3 (1:1000), anti-ATG7 (1:1000) and anti-LC3A/B (1:1000) from Cell Signaling Technology (Danvers, MA); anti-CHOP, anti-p62 (1:500, Santa Cruz Biotechnology), and anti-α Tubulin (1:5000, Abcam) for overnight at 4˚C. Anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) was used at a dilution of 1:5000 for 1 hr. at room temperature. The binding of specific antibodies was visualized via exposure to a photographic film after treating with enhanced chemiluminescence system reagents (Fisher Scientific, USA). The film was scanned and the band density was quantified by ImageJ (NIH) software. The results were expressed as a relative ratio of the target protein to reference protein.
6
Western Blot Analysis of Autophagy Markers
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Cell pellets were lysed by lysis buffer and subjected for analysis. The samples were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Merck Millipore). After blocked with 5% non-fat dry milk and immunoblotted with the appropriate primary and secondary antibody, the blot were measured by using the Fusion FX7 ECL western blot system (Vilber Lourmat). Antibodies used are as follows:
Anti-LC3 A/B (#12741), Anti-SQSTM1/p62 (#39749), Anti-ATG3 (#3415), Anti-ATG5 (#12994), Anti-ATG7 (#8558), Anti-ATG12 (#4180), Anti- β-actin (#3700) from Cell Signaling Technology, Anti-ISG15 (A1182) from ABclonal, Anti-FlAG (F3165) and Anti-HA (H6908) from Sigma Aldrich, and Anti-ISG15 (F-9) (sc-166755) used for ISGylation was from Santa Cruz Biotechnology.
Anti-LC3 A/B (#12741), Anti-SQSTM1/p62 (#39749), Anti-ATG3 (#3415), Anti-ATG5 (#12994), Anti-ATG7 (#8558), Anti-ATG12 (#4180), Anti- β-actin (#3700) from Cell Signaling Technology, Anti-ISG15 (A1182) from ABclonal, Anti-FlAG (F3165) and Anti-HA (H6908) from Sigma Aldrich, and Anti-ISG15 (F-9) (sc-166755) used for ISGylation was from Santa Cruz Biotechnology.
7
Comprehensive Protein Expression Analysis in Cell Lysis
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Cells were lysed with cell lysis buffer. Equal amounts of total proteins were loaded onto 4-12% SDS–PAGE gels and transferred to PVDF membranes (GE Health care Life Sciences, Marlborough, MA, USA). The membranes were blocked with 5% skim milk in TBST buffer and incubated with primary antibodies - anti-DNMT1 (Abcam, Cambridge, UK), anti-cleaved caspase 3, anti-E-cadherin, anti-cleaved caspase 9, anti-cleaved PARP1, anti-ATG3, anti-ATG5, anti-ATG7, anti-ATG12, anti-ATG16L1 (Cell Signaling Technology, Denvers, MA, USA), anti-LC3A (Novus Biologicals, Littleton, CO, USA), anti-Beclin (BD Biosciences), and anti-β-actin (Protein Tech group, Rosemont, IL, USA) overnight at 4°C. The membranes were subsequently incubated with specific horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized by using a AI600 system (GE Healthcare, Marlborough, MA, USA).
8
Western Blotting of Autophagy Proteins
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Protein expression in cells was measured by immunoblotting analysis as previously described [22 (link)]. Briefly, total protein from MIN6 cells was prepared using radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with protease and phosphatase inhibitors (Thermo Fisher Scientific). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The following primary antibodies were used: anti-LC3, anti-β-actin, anti-Atg3, anti-Atg5, anti-Atg7, anti-SQSTM1/p62, anti-beclin1, anti-phosphor-mammalian target of rapamycin (mTOR), anti-Unc51 like autophagy activating kinase (ULK) 1, anti-cleaved caspase-3, and anti-total caspase-3 (Cell Signaling Technology, Danvers, MA, USA).
9
Autophagy Regulation Mechanism Elucidation
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The LN18 and HEK293T cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. The anti-NOP53 rabbit polyclonal antibody was purified as previously described [16 (link)]. The anti-LC3B, anti-H3, anti-phospho-H3, anti-ATG12, anti-ATG7, and anti-ATG3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-ZKSCAN3 and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Unless otherwise specified, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
Molecular Mechanisms of Cystathionine Metabolism
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