Mouse ileal epithelial cells were collected by scraping from mouse ileum as previously described.65 (link) Briefly, mouse epithelial cells were lysed in lysis buffer (1% Triton X-100 (Sigma-Aldrich, X100), 150 mM NaCl ( J.T.Baker 3624-19), 10 mM Tris ( Fisher Scientific, BP152-5) pH 7.4, 1 mM EDTA(Fisher Scientific, BP120-1), 1 mM EGTA(Sigma-Aldrich, E3889) pH 8.0, 0.2 mM sodium ortho-vanadate (Sigma-Aldrich, S6508), and protease inhibitor cocktail (Roche Diagnostics, 118367001)). Cultured cells were rinsed twice in ice-cold HBSS (Sigma-Aldrich, H1387), lysed in protein loading buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol (Amresco, 0281), 2% SDS (Sigma-Aldrich, L3771), 0.1% bromophenol blue (IBI Scientific, IB74040), and 10% glycerol (Sigma-Aldrich, G5516)), and sonicated (Branson Sonifier, 250). Equal amount of protein was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (Bio-rad, 162-0112), and immunoblotted with primary antibodies: VDR (Santa Cruz, sc13133), Beclin-1(Santa Cruz, sc10086), Villin (Santa Cruz, sc7672), LC3B (Cell Signal Technology Inc., 2775), ATG16L1 (Abgent, AP18176), p62 (Abgent, AP2183B) or β-actin (Sigma-Aldrich,A1978) antibodies and visualized by ECL chemiluminescence (Thermo Scientific, 32106).
Beclin 1
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Beclin-1 is a gene that encodes a protein involved in the regulation of autophagy, a cellular process that recycles damaged or unnecessary components within the cell. The Beclin-1 protein plays a key role in the initiation and formation of autophagosomes, which are the vesicles that engulf the cellular material to be degraded and recycled. Beclin-1 is considered a critical component of the autophagy pathway and is widely studied in the context of various biological processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (26 mentions)
Bcl 2, by Santa Cruz Biotechnology (20 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (11 mentions)
Gapdh, by Santa Cruz Biotechnology (10 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Bafilomycin a1, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Caspase 9, by Santa Cruz Biotechnology (5 mentions)
Caspase 3, by Santa Cruz Biotechnology (5 mentions)
Lc3 1 2, by Santa Cruz Biotechnology (5 mentions)
Rapamycin, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
β actin, by Merck Group (4 mentions)
Lc3 2, by Santa Cruz Biotechnology (4 mentions)
Cleaved caspase 3, by Santa Cruz Biotechnology (4 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
Z vad fmk, by Merck Group (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Cytochrome c, by Santa Cruz Biotechnology (3 mentions)
P akt, by Santa Cruz Biotechnology (3 mentions)
108 protocols using beclin 1
1
Isolation and Analysis of Mouse Ileal Epithelial Cells
2
Apoptosis pathway protein analysis
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3
Protein Expression Analysis in Cardiac Tissues
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The homogenized ventricles tissue or cultured cells were incubated in lysis buffer (pH 7.4) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 1% Triton-X 100, 1% NP-40, 0.1 mM Na3VO4, 12.5 mM b-glycerophosphate, 1 mM phenylmethanesulfonylfluoride (PMSF) and complete protease inhibitor cocktail. Insoluble heart tissues or cell fractions were removed by centrifugation at 13,000 × g, 4°C for 30 min. The concentration of the extracted proteins was measured by bicinchonininc acid assay (Sigma-Aldrich, St. Louis, MO, USA). The extracted proteins were boiled for 3–5 min. in 5× loading buffer. The expression of AT1 receptor, LC3b-II, beclin-1 and phosphorylation of Akt (p-Akt) were detected by immune-blotting with antibodies against AT1 receptor (catalogue no.sc-1173G; Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3b-II (catalogue no. 2775; Cell Signaling Technology, Danvers, MA, USA), beclin-1 (catalogue no. 3495; Cell Signaling Technology), and p-Akt (catalogue no. 9275; Cell Signaling Technology), and detection was performed by using an ECL Western-blotting Detection Reagents (RPN2106; GE Healthcare, Little Chalfont, Buckinghamshire, UK) visualized densitometry with LAS-300 Image software.
4
Scutellarin Ameliorates Diabetic Neuropathy
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Scutellarin (purity 98%) was purchased from Honghe Qianshan Bioengineering Co., Ltd. (Honghe, China). Streptozocin (STZ) and rosiglitazone were obtained from Multi Sciences (Lianke) Biotech, Co., Ltd. (Hangzhou, China). Antibodies to detect β-actin, procaspase-3, procaspase-8, procaspase-9, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Cyt-C, Bcl-2, Beclin-1, and LC3-II were ordered from Santa Cruz Biotechnology. The procaspase-12 polyclonal antibody was ordered from Abcam Company. Adult male Sprague-Dawley (SD) rats (180 to 200 g) were obtained from the Experimental Animal Center of Kunming Medical University, China. Two weeks after the adaptive feeding of the rats, they were randomly divided into control group, model group, SCU low-dose (100 mg/kg/d), high-dose (200 mg/kg/d) treatment group, and rosiglitazone (5 mg/kg/d) positive control group (each group of ten). The model group was given a high-sugar and high-fat diet for 8 weeks and injected with STZ by 35 mg/kg intraperitoneally, while the control group was given a normal diet. The SCU groups were administered orally for 8 weeks [12 (link)].
5
Immunoblotting Analysis of Cellular Proteins
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Rabbit antibodies against GADD153, GRP78, Beclin-1, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), while those against LC-3 were purchased from MBL. Secondary horseradish peroxidase- (HRP-) conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Cells were lysed in a buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM phenylmethylsulfonyl fluoride, 8 μg/ml aprotinin, and 2 μg/ml leupeptin (pH 7.4). For immunoblotting, proteins resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA), which were then exposed to primary and then secondary antibodies. Chemiluminescence detection was performed with an ECL™ Western Blotting Detection Kit (Amersham). The signal intensities of the corresponding bands were measured by a Light Capture equipped with CS Analyzer software (ATTO, Osaka, Japan).
6
Alisol A 24-acetate Induces Autophagy in Renal Cells
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Trypsin was provided by KeyGEN Biotech Co., Ltd (Nanjing, China). Dulbecco’s modified eagle medium (DMEM)-F12 (1:1) medium with high-glucose and fetal bovine serum (FBS) were purchased from Gibco/BRL (Grand Island, New York, NY, USA). 3-4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H-tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), and 3-methyl adenine (3-MA) were provided by Sigma Chemical (St. Louis, MO, USA). Alisol A 24-acetate (24A) and Alisol B 23-acetate (23B; purity ≥95%) were purchased from Tianjin Evans Science and Technology Co., Ltd (Tianjin, China). Primary and second antibodies against LC3, Beclin-1, Bcl-2, Bcl-xl, Clusterin, Kim-1, TFF-3 and β-actin were provided by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Hematoxylin-eosin staining solution was offered by Shanghai Yuanmu Biotechnology Co., Ltd (Shanghai, China). EliVision plus and DAB kits were offered by MXB Biological Technology Co., Ltd (Fuzhou, Fujian, China). Monopotassium phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride were obtained from Nanjing Nanao Science and Technology Co., Ltd (Nanjing, China).
7
Beclin 1 Knockdown and Resveratrol Treatment
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Beclin 1 (catalog no. sc-29797) and scrambled control siRNA (catalog no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. The Beclin 1 siRNA sequences were as follows: Forward, 5′-CAGCUCAACGUCACUGAAATT-3′ and reverse, 5′-UUUCAGUGACGUUGAGCUGTT-3′. The control siRNA sequences were not available. Briefly, 100 nM of Beclin 1 or control siRNA was transfected into cells using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and non-transfected cells were set as a blank control. After 24 h, cells were treated with 40 µM Res for an additional 48 h and subsequently analyzed via western blotting as previously described.
8
Apoptosis Induction in SW620 Colon Cancer Cells
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SW620 human colon cancer cells from the American type culture collection (Rockville, MD, USA) were cultured in RPMI 1640 medium (Invitrogen Corp, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 1 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. As4O6 was obtained from Chonjisan institute (Seoul, Korea). Antibodies against procaspase 3, poly (ADP-ribose) polymerase (PARP), β-catenin, DR4, DR5, Bax, Bcl-2, Bid, cyclin B1, XIAP, p21, AKT 1/2/3 (H-136), phospho-Akt (Ser473), ERK, phospho-ERK (E-4), p53 and Beclin 1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against LC3, and Beclin-1, were purchased from PharMingen (San Diego, CA, U.S.A.). Antibodies against phospho-Akt (Thr 308), procaspase 8, procaspase 9, phospho-p38 MAPK, cdc2, JNK, and phospho-JNK were purchased from Cell signaling Technology, Inc. (Beverly, MA, USA). Antibody against β-actin was purchased from Sigma (Beverly, MA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin, and an enhanced chemiluminescence (ECL) kit were purchased from Amersham (Arlington Heights, IL, USA). All other chemicals not specifically cited here were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
9
Autophagy Modulation in Cancer Therapeutics
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Metformin (D150959), CQ (C6628), MDC (30432), 3-MA (M9281), 4',6-diamidino-2- phenylindole (DAPI; D9542), antibodies against anti-β and LC3, anti-rabbit secondary antibody, anti-mouse secondary antibody and Cy3-labeled rabbit secondary antibody were purchased from Sigma (St. Louis, MO, USA). Antibodies against p-Stat3 (Y705), Stat3, cyclin D1, LC3, PCNA and PARP were purchased from Cell Signaling (Beverly, MA, USA); antibodies against p-AMPK, p-mTOR, Beclin-1, Bcl-2, p62 and Bax were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-phospho-Histone H3 (Ser10) antibody was purchased from Upstate (Millipore, Billerica, MA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were obtained from Gibco (Life Technologies Gibco/BRL, New York, NY, USA). Crystal violet staining and JC-1 were obtained from Beyotime Institute of Biotechnology (Shanghai, China). ApopTag plus peroxidase in situ apoptosis detection kit was obtained from Millipore. AO was obtained from Poly-Sciences (Warrington, PA, USA). Stat3, AMPK, Beclin-1 and Atg5 siRNAs were purchased from Shanghai GenePharma (Shanghai, China). Lipofectamine 2000 was bought from Invitrogen (Carlsbad, CA, USA).
10
Western Blotting of Cortex and Myocardium Proteins
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Western blotting with proteins of the cortex and myocardium was performed as described previously (Mlyniec et al. 2014a , b (link)). To confirm equal loading of the samples on the gel, the membranes were re-probed with an antibody specific to GAPDH as an internal control. The specific primary antibodies used included rabbit polyclonal antibodies against CAIII (1:1000; abcam), Beclin-1 (1:200; Santa Cruz), LC3 (1:1000; abway), P53 (1:1000; abcam), IL-17 (1:1000; abcam), NF-κB (1:200; Santa Cruz), GAPDH (1:2000; Boster). Finally, the X-ray films were developed and fixed in a dark room.
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