Hypoxia Incubator Chamber (Stemcell Technologies, Vancouver, Canada 27310), Single Flow Meter (Stemcell Technologies, 27311), hypoxic gas (3% O2, 5% CO2, and 92% N2, Beijing Yongsheng Gas Technology Limited Company, Beijing, China), H2O2 (Sigma-Aldrich, St. Louis, MO, USA H1009), rapamycin (Sigma-Aldrich, V900930), 3-methyxanthine (3-MA; Sigma-Aldrich, M9281), anti-β-actin antibody (TransGen Biotech, Beijing, China HC201-01), anti-LC3B antibody (Cell Signaling Technology, Inc., Danvers, MA, USA 3868), anti-Beclin 1 antibody (Cell Signaling, 3495), anti-p53 antibody (Cell Signaling, 2524), IRDye® 800CW Goat anti-rabbit IgG (LI-COR, Lincoln, NE, USA 926-32211), IRDye® 800CW Goat anti-mouse IgG (LI-COR, 926-32210), CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega Corporation, Madison, WI, USA G7571), Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA 11995073), monodansylcadaverine (MDC; Sigma-Aldrich, D4008), and ROS Assay Kit (Beyotime, Shanghai, China S0033) were used.
Anti beclin 1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Beclin 1 antibody is a laboratory reagent used to detect the Beclin 1 protein in biological samples. Beclin 1 is a key regulator of autophagy, a cellular process involved in the degradation and recycling of cellular components. This antibody can be used in various techniques, such as western blotting, immunohistochemistry, and immunofluorescence, to study the expression and localization of Beclin 1 in different cell types and experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Santa Cruz Biotechnology (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Anti p62 antibody, by Cell Signaling Technology (2 mentions)
Alexa 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent hrp substrate, by Merck Group (2 mentions)
Anti synaptophysin antibody, by Cell Signaling Technology (2 mentions)
Anti nr2a antibody, by Cell Signaling Technology (2 mentions)
Anti gabaar antibody, by Cell Signaling Technology (2 mentions)
Anti nr2b antibody, by Cell Signaling Technology (2 mentions)
Anti psd 95 antibody, by Cell Signaling Technology (2 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Hypoxia incubator chamber, by STEMCELL (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Anti lc3b antibody, by Cell Signaling Technology (1 mentions)
Anti p53 antibody, by Cell Signaling Technology (1 mentions)
Rapamycin, by Merck Group (1 mentions)
13 protocols using anti beclin 1 antibody
1
Hypoxia-Induced Autophagy Modulation
2
Osteogenic Differentiation and Autophagy
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α-Modification minimum essential medium (α-MEM) was purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Fetal bovine serum (FBS) was acquired from Clark Bioscience (Claymont, DE, USA). The MTT Cell Proliferation and Cytotoxicity Assay kit was purchased from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). The Alkaline Phosphatase (ALP) Assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). VK2, vitamin C (VC), and dexamethasone (Dex) were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). β-Glycerophosphate and alizarin red were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The autophagy inhibitor 3-methyladenine (3-MA) and agonist rapamycin were purchased from Selleck Chemicals (Houston, TX, USA). Rabbit anti- microtubule associated protein 1 light chain 3-α (LC3)b antibody was purchased from Abcam (Cambridge, UK; cat. no. ab192890). Rabbit anti-β-actin antibody and goat anti-rabbit IgG antibody were purchased from BIOSS (Beijing, China; cat. nos. bs-0061R and bs-0295G, respectively). Anti-beclin 1 antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. no. 3738S) and Cy3-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) was obtained from Wuhan Sanying Biotechnology (Wuhan, China; cat. no. SA00009-2).
3
ApoG2 Anticancer Activity in CNE-2 Cells
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The human NPC CNE-2 cell line was obtained from the Cancer Institute of the Southern Medical University (Guangzhou, China). ApoG2 was provided by the University of Michigan (Michigan, USA). An ApoG2 stock solution was freshly prepared at a concentration of 20 mmol/l in 100% dimethyl sulfoxide (DMSO) on the day of the experiment, and then diluted to the specific concentrations (0, 5, 10, 20, 40, 60 and 80 µmol/l) required for a particular study.
Control groups in the experiments were treated with 0.1% DMSO alone. All primary antibodies used in western blot analysis were rabbit anti-human monoclonal antibodies. The anti-Bcl-2 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; sc-492; dilution 1:1,000), the anti-beclin-1 antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; 3495; dilution, 1:1,000) and the anti-β-actin antibody was purchased from Abmart Biomedical (Shanghai, China; P30002; dilution, 1:2,000). The secondary antibodies were biotin-labeled goat anti-rabbit antibodies purchased from Boster Biotechnology Inc. (Wuhan, China; BA1003; dilution, 1:400). A pre-stained protein ladder was purchased from Thermo Fisher Scientific, Inc., Waltham, MA, USA), and other reagents and laboratory supplies were purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA).
Control groups in the experiments were treated with 0.1% DMSO alone. All primary antibodies used in western blot analysis were rabbit anti-human monoclonal antibodies. The anti-Bcl-2 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; sc-492; dilution 1:1,000), the anti-beclin-1 antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; 3495; dilution, 1:1,000) and the anti-β-actin antibody was purchased from Abmart Biomedical (Shanghai, China; P30002; dilution, 1:2,000). The secondary antibodies were biotin-labeled goat anti-rabbit antibodies purchased from Boster Biotechnology Inc. (Wuhan, China; BA1003; dilution, 1:400). A pre-stained protein ladder was purchased from Thermo Fisher Scientific, Inc., Waltham, MA, USA), and other reagents and laboratory supplies were purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA).
4
Protein Extraction and Western Blot Analysis
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The extraction of total protein from the treated cells was carried out using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Nantong, China). The concentration of protein was quantified with bicinchoninic acid (BCA) protein measurement kit (Thermo Fisher Scientific, USA). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The membrane was blocked with 5% skim milk at room temperature for 2 h, and then incubated with anti-GRP78 antibody, anti-PERK antibody, anti-Beclin1 antibody, anti-LC3 I/II antibody, and anti-GAPDH antibody (Cell Signaling Technology, USA) at 4 °C overnight. Next day, the relative secondary antibody was added, and the membrane was incubated at room temperature for 1 h. Then, enhanced chemiluminescence (ECL) reagent was added, and the membranes were detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
5
Protein Expression Analysis by Western Blot
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The total tumour cells were homogenized and the total protein was extracted by RIPA buffer. And then the extraction and detection of the proteins were performed by Western blotting. Anti-HDGF antibody (1:1000), anti-LC3 I, LC3 II antibody (1:1000), anti-Beclin-1 antibody (1:1000), anti-P62 antibody (1:1000), Anti-HDGF antibody (1:1000), anti-GAPDH antibody (1:2000) (Cell Signaling Technology, Boston, USA), then membranes were incubated with a secondary antibody (1:5000, Cell Signaling Technology, USA), and all protein samples were measured using a chemiluminescence (Bio-Rad, USA) imaging system.
6
Synaptic Protein Immunodetection
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Anti-Synaptophysin antibody, anti-PSD-95 antibody, anti-NR2B antibody, anti-NR2A antibody, anti-GABAAR antibody, Anti-Beclin-1 antibody was bought from Cell Signaling Technology (MA, USA). Anti-β-actin antibody was purchased from Santa Cruz Biotechnology, Inc. CA (California, USA). The Alexa 488-conjugated goat anti-rabbit IgG was bought from Invitrogen (San Diego, USA).
The chemiluminescent HRP substrate was purchased from Millipore Corporation (MA, USA).
The chemiluminescent HRP substrate was purchased from Millipore Corporation (MA, USA).
7
Immunohistochemical Analysis of Beclin1 Expression
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Paraffin-embedded liver tissue wax blocks were sectioned at 5 μm. After being dewaxed and rehydrated, the sections were incubated in 3% hydrogen peroxide/methanol. Heat-induced antigen retrieval was performed by heating in 10 mM sodium citrate buffer for 10 min. Sections were incubated in anti-Beclin1 antibody (Cell Signaling Technology) at 1:200 dilution at 4 °C overnight. 3,3′-Diaminobenzidine Substrate Chromogen System (Dako, Carpinteria, CA, USA) was employed during the detection procedure. Subsequently, the sections were counterstained using hematoxylin for 10 s. Finally, after being dehydrated in ethanol, cleared in xylene and mounted, the sections were observed in the light microscope by a pathologist who was initially blinded to treatment groups, and five random fields of each slide were semi-quantified and averaged using the software ImageJ 1.48 (National Institutes of Health, Bethesda, MD, USA) according to its instructions, then come up with the data of density of target protein positive cell, and the relative density (/sham) of protein represents the protein expression level of HO-1.
8
Immunoblotting Protocols for Autophagy Regulators
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The primary antibodies used for immunoblotting were anti-LC3 antibody (Cell Signaling Technology 4108), anti-GAPDH antibody (Millipore MAB374), anti- NRF2 antibody (GeneTex GTX103322), anti-SQSTM1 antibody (GeneTex GTX100685), and anti-Beclin 1 antibody (Cell Signaling Technology 3495). The secondary antibodies used for immunoblotting were anti-mouse IgG (whole molecule)-peroxidase antibody produced in rabbit (Sigma-Aldrich A9044) and anti-rabbit IgG (whole molecule)-peroxidase antibody produced in goat (Sigma-Aldrich A0545). The pEGFP-LC3 plasmid was a gift from Dr. Tamotsu Yoshimori, Department of Genetics, Graduate School of Medicine, Osaka University. LPS-EB (LPS from E. coli O111:B4) was purchased from InvivoGen (CA, USA). 3-methyladenine and chloroquine diphosphate salt were from Sigma‐Aldrich. Bay 11–7082 was from Calbiochem (Sigma-Aldrich).
9
Wogonin Attenuates Diabetic Kidney Injury
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Wogonin was acquired from Aladdin Biology Technology Institute (W101155, CAS 632–85-9, Shanghai, China). Streptozotocin (STZ) was obtained from Sigma-Aldrich (Saint Louis, MO, USA). PI3K inhibitor LY294002 was acquired from MedChem Express (Princeton, USA). DMEM was obtained from Gibco (Gibco, CA). The NF-κB p65 and p-NF-κB p65 antibody, anti-LC3 antibody, anti-P62 antibody, anti-Beclin1 antibody, anti-Akt and anti-p-Akt were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies anti-KIM-1, anti-Atg7 and anti-fibronectin (FN) were obtained from Abcam (Cambridge, USA). Anti-α-SMA and anti-E-cadherin antibodies were acquired from Proteintech (Wuhan, China). Anti-Col-I antibody was acquired from Bioss (Shanghai, China). Anti-PI3K and Anti-p-PI3K were obtained from Affinity Biosciences (Affinity, USA). Periodic acid-Schiff (PAS), creatinine (CRE), blood urea nitrogen (BUN) kits were purchased from Jiancheng Biology Technology Institute (Nanjing, China). Albumin ELISA kit was acquired from Abcam (Cambridge, USA).
10
Immunohistochemistry Analysis of Autophagy Markers
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Paraffin-embedded sections of lungs (4% phosphate-buffered and formalin-fixed for 24 h at room temperature; 4-µm thick) were dewaxed in xylene, rehydrated in ethanol and blocked with 3% BSA (cat. no. G5001; Wuhan Servicebio Technology Co., Ltd.) for 30 min at room temperature. Following antigen retrieval, sections were incubated with anti-LC3B antibody (1:300; cat. no. 14600-1-AP; ProteinTech Group, Inc.), anti-SQSTM1/p62 antibody (1:400; cat. no. 88588S; Cell Signaling Technology, Inc.) and anti-Beclin 1 antibody (1:400; cat. no. 11306-1-AP; ProteinTech Group, Inc.) for 12 h at 4°C. Then, sections were incubated with HRP-conjugated secondary antibodies (1:200; cat. no. GB23303; Wuhan Servicebio Technology Co., Ltd.) for 50 min at 24°C. Cells were counted in five randomly selected fields using an optical microscope (magnification, ×400) and analysis was performed using ImageJ 1.53 software (National Institutes of Health), as previously described (41 (link)).
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