Equal amounts of total protein from colon tissue or cultured cells were separated by SDS-PAGE and then transferred to nitrocellulose membranes by semi-dry blotting as previously described[16 (link),19 (link),20 (link)]. After blocking the membranes with 5% non-fat dry milk, they were probed with antibodies to either ATG16L1 (Cell Signaling, Beverly, MA, United States), NOD2 (Cayman Chemical, Ann Arbor, MI, United States), LC3B (Cell Signaling) or total GAPDH (Santa Cruz Biotechnology, Dallas, TX, United States). After washes, the membranes were incubated with appropriate horseradish peroxidase-associated secondary antibodies before signals were visualized with the enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, United States).
Atg16l1
Manufactured by Cell Signaling Technology
Sourced in United States
ATG16L1 is a protein that plays a crucial role in the autophagy process. It is a key component of the ATG16L1 complex, which is involved in the formation of autophagosomes, the double-membrane vesicles that engulf and transport cellular materials to the lysosome for degradation. The ATG16L1 protein acts as a scaffold, helping to recruit other autophagy-related proteins to the site of autophagosome formation.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Cell Signaling Technology (10 mentions)
Atg12, by Cell Signaling Technology (3 mentions)
Lc3 1 2, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Proteintech (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Atg14, by Cell Signaling Technology (2 mentions)
Atg9a, by Cell Signaling Technology (2 mentions)
Fip200, by Cell Signaling Technology (2 mentions)
P mtor, by Cell Signaling Technology (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence detection system, by Cytiva (1 mentions)
Ab182032, by Abcam (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab32618, by Abcam (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab13556, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Fudebio (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Phospho s6, by Cell Signaling Technology (1 mentions)
24 protocols using atg16l1
1
Western Blot Analysis of Autophagy Regulators
2
Western Blot Analysis of Autophagy Markers
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Whole-cell lysates were prepared as previously described [6 (link), 7 (link)]. Equal amounts of protein (10–50 μg) were resolved by 12% SDS–PAGE. After electrophoresis, separated proteins were transferred onto nitrocellulose membrane. The membrane was blocked in 5% non-fat milk, followed by overnight incubation with primary antibodies. After incubation with HRP-conjugated secondary antibody, the positive immune reactive signal was detected by ECL (Fude Biotech, Hangzhou, China). Antibodies specific for GST (sc-459, 1:500), CD14 (sc-9150, 1:500), and β-actin (sc-47778, 1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for GRP78 (ab32618, 1:1000), CD14 (ab182032, 1:1000), and TLR4 (ab13556, 1:1000; ab22048, 1:1000) were obtained from Abcam (Cambridge, UK). Antibodies specific for Atg3 (3415, 1:1000), Atg5 (12994, 1:1000), Atg7 (8558, 1:1000), Atg12 (4180, 1:1000), Atg16L1 (8089, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies specific for Caspase 3 (19677-1-AP, 1:1000), p62 (18420-1-AP, 1:1000) were purchased from Proteintech. Anti-LC3 (PM036, 1:1000) was purchased from MBL life science.
3
Western Blot Analysis of Cell Signaling Proteins
4
Autophagy and Apoptosis Biomarkers
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Cell lysates were extracted from siRNA-treated cells at 0–2 days of incubation, washed twice with 1× PBS, and resuspended in radioimmunoprecipitation assay (RIPA) buffer (71009; Merck, Darmstadt, Germany). Protein expression was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. Equal amounts of protein samples were separated using SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membranes (Merck Millipore Life Science). The membranes were blocked for 3 h at room temperature with 5% non-fat milk in PBS with Tween 20 and incubated with primary antibodies at 4°C overnight. Primary antibodies were against Beclin, LC3, Atg5, and Atg16L1, respectively (1:1,000; Cell Signaling Technology, Danvers, MA, USA), p53 (1:200; Abcam, Cambridge, UK), survivin (1:1,000; Abcam), P-gp (1:500; GeneTex, Irvine, CA, USA), ABCC10 (1:200; Abcam), and GAPDH (1:10,000; GeneTex). The signals of immunoreactive blots were detected using the Enhanced Chemi-Luminescence (ECL) western blotting reagent (Perkin-Elmer, Waltham, MA, USA).
5
Western Blot Analysis of Autophagy Markers
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Ischemic tissues and cells were kept in ice-cold RIPA buffer with 0.1 mM phenylmethylsulfonylfluoride (PMSF) (Sigma, St. Louis, MO, USA) to extract the total proteins. The resolved proteins were transferred to polyvinylidene fluoride (PVDF) membrane under the condition of 300 mA for 1.5 h, and then incubated at room temperature for 2 hours in a TBST buffer (20 mM Tris-HCl, pH 7.6,150 mM NaCl, 0.1% Tween-20) containing 5% no-fat milk. Specific primary antibodies were incubated at 4°C overnight, including ATG16L1 (Cell Signaling Technology, 8089S), SQSTM1/p62 (Servicebio, GB11531), GAPDH (Proteintech, 60004-1-Ig) and LC3A/B (Servicebio, GB11124). Further, the PVDF membrane was incubated with corresponding HRP-conjugated secondary antibodies for 90 min at room temperature. Blots were visualized by an enhanced chemiluminescent (ECL) reagent (Amersham Biosciences, Piscataway, USA) under a Bio-Rad ChemiDoc MP system (Bio-Rad, Hercules, CA, USA). Bands were quantified through Image J.
6
Western Blot Quantification Protocol
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Equal amounts of total protein were separated by SDS-PAGE and then transferred to nitrocellulose membranes by semi-dry blotting as previously described [18 (link), 19 (link)]. After blocking the membranes with 5% non-fat dry milk, they were probed with antibodies to ATG16L1, Beclin-1 and LC3B (Cell Signaling, Beverly, MA) and then developed with HRP-conjugated second antibodies (Zymed Laboratories, San Francisco, CA) and enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL). Capture the chemiluminescent signals using a camera-based imager and use image analysis software to read the band density of the target protein.
7
Analyzing Membrane Trafficking Pathways
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The chemicals used in this study were: MBCD (Sigma, C4555), cholesterol-water soluble (Sigma, C4951), Baf A1 (Santa Cruz, CAS 88899-55-2), Wort (Santa Cruz, CAS 19545-26-7), cholera toxin subunit B conjugated with Alexa Fluor 594 (CTxB, Invitrogen, C34777), DAB (Dako, K401011), NEM (Sigma, E3876), DTT (Sigma, D9779), human transferrin peroxidase (Rockland antibodies and assays, 009-0334). The antibodies used were: anti-MAP1LC3B/LC3B (Sigma, L7543), anti-ACTB/β-actin (Sigma, A5441), anti-TFRC/TFRC (Invitrogen, 136,800), anti-VAMP3 (Santa Cruz, sc-514843), anti-CAV1/caveolin-1 (BD Pharmingen, 610,060), anti-ATG9A (Abcam, ab108338), anti-RAB11 (Cell Signaling Technology, 5589), mouse anti-STX6 (Invitrogen, 701,823), WIPI2 (Abcam, ab105459), and ATG16L1 (Cell Signaling Technology, 8089).
8
Protein Extraction and Western Blot Analysis
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Protein extraction and western blot analysis were performed as described by Yang et al. [24 (link)]. The following primary antibodies were used: CLDN6 (V118, 1:1000) (Bioworld Technology, USA); ERα (ab66102, 1:1000), ERβ (ab288, 1:1000) (Abcam, Cambridge, UK); LC3B (D11, 1:1000), beclin1 (D40C5, 1:1000), Atg5 (D5F5U, 1:1000), Atg16L1 (D6D5, 1:1000) (Cell Signaling Technology, Danvers, MA, USA); ZO-1 (21773–1-AP, 1:1000), UVRAG (19571–1-AP, 1:1000), β-actin (60008–1-Ig, 1:5000) (Proteintech Group, USA). The signals were visualized using ECL reagent (Millipore, Billerica, MA, USA).
9
Immunoblot Analysis of Signaling Proteins
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Equal amounts of total protein were separated by SDS-PAGE and then transferred to nitrocellulose membranes by semi-dry blotting as previously described [23 (link),24 (link)]. After blocking the membranes with 5% non-fat dry milk, they were probed with antibodies to either phosphorylated p38, JNK, Akt (Cell Signaling, Beverly, MA), phosphorylated ERK (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated IκB (New England BioLabs, Beverly, MA), anti-MyD88, ATG16L1, Beclin-1, Atg5, rabbit anti-LC3 (Cell Signaling, Beverly, MA), or anti-NOD1 and NOD2 (Cayman Chemical, Ann Arbor, MI), and then developed with horseradish peroxidase-conjugated second antibodies (Zymed Laboratories, San Francisco, CA) and enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL). Appropriate exposures to X-ray film were made, and the filters then stripped and re-probed with antibodies to GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) as appropriate.
10
Modulation of Apoptosis and Autophagy Pathways by Thymoquinone
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The cells were treated with TQ for 24 h, and the treated cells were lysed using a cold mammalian protein extraction buffer kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) with protease inhibitor cocktails for 20 min to prepare the total cell lysates. The samples were separated in a 12.5% polyacrylamide gel and then transferred onto a nitrocellulose membrane. Afterward, the membranes were blocked in 5% non-fat milk in Tris-buffered saline with Tween buffer (20 mM Tris, 137 mM NaCl, pH 7.6, 0.1% Tween-20) for 1 h. The membranes were then probed with antibodies specific for caspase-9, caspase-8, Bax, Bcl-2, Bid (Santa Cruz Biotechnology Inc. California, USA), p-histone H2A.X, caspase-3 (Millipore Corp., Bedford, MA; Chemicon International, Inc., Temecula, CA), Rubicon, PI3K Class III, Becline, Atg14, Atg7, Atg16L1, Atg5, Atg12, LC3A, p62, PARP and mToR (Cell Signaling Technology Inc., Danvers, MA), along with appropriate peroxidase-conjugated secondary antibodies. The signal was subsequently detected using an ECL commercial kit, and relative photographic density was quantified using an ImageQuant LAS 4000 mini (GE Healthcare, Little Chalfont, Buckinghamshire, UK) [19] (link).
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