Ischemic cerebrum cells and tissues were lysed using a RIPA Lysis Buffer. Total protein was extracted using a Total Protein Extraction Kit (Takara) according to the manufacturer's instructions, and the quality was detected using the Bradford method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein extracts. An equal amount of protein was transferred onto a polyvinylidene fluoride membrane, and incubated with primary antibodies (anti-p-mTOR, anti-Beclin-1, anti-Mcl, 1:500, [Sigma, USA]) or β-actin (1:500, [R&D, China]) at 4°C for 24 h. The membranes were then incubated with the secondary antibody (1:1000) for another 2 h at room temperature. The ECL method and Image J software were used to visualize the bands. The antibodies were purchased from Abcam (UK). β-actin acted as an internal control.
Anti beclin1
Manufactured by Merck Group
Sourced in United States
Anti-Beclin1 is a lab equipment product that targets the Beclin-1 protein. Beclin-1 is a key regulator of autophagy, a cellular process involved in the degradation and recycling of damaged organelles and proteins. Anti-Beclin1 is used to study the role of Beclin-1 in various cellular processes and pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3, by Merck Group (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti lc3, by Thermo Fisher Scientific (2 mentions)
Anti galectin 3, by Santa Cruz Biotechnology (2 mentions)
Anti lamp1, by Developmental Studies Hybridoma Bank (2 mentions)
Anti c albicans, by GeneTex (2 mentions)
Anti goat secondary antibodies conjugated with alexa fluor 488 and horseradish peroxidase hrp, by Thermo Fisher Scientific (2 mentions)
Llome l7393, by Merck Group (2 mentions)
3 ma m9281 500mg, by Merck Group (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Annexin 5 alexa fluor 488 ready flow conjugate, by Thermo Fisher Scientific (2 mentions)
Anti p62, by Cell Signaling Technology (2 mentions)
Anti alix, by Santa Cruz Biotechnology (2 mentions)
Anti tsg101, by Abcam (2 mentions)
Total protein extraction kit, by Takara Bio (1 mentions)
β actin, by R&D Systems (1 mentions)
Anti p mtor, by Merck Group (1 mentions)
Non fat milk, by Merck Group (1 mentions)
Non fat milk, by Bio-Rad (1 mentions)
Hyperfilm ecl, by Cytiva (1 mentions)
10 protocols using anti beclin1
1
Ischemic Cerebrum Protein Expression
2
Western Blot Analysis of EGFR and Beclin1
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Tissue samples were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors, following standard procedures. After protein determination, using BioRad protein assay (BioRad, Milan, Italy), equal amounts of proteins (40 μg) were resolved on 8% SDS-PAGE gel and transferred to a nitrocellulose membrane (BioRad). Membranes were blocked with 3% nonfat milk (BioRad) in PBS Tween 0.05% (PBST) and incubated overnight at 4°C with the following antibodies: anti-EGFR (undiluted, Novocastra), anti-Beclin1 (1 : 270, Sigma-Aldrich), and anti-β-actin (1 : 500, catalogue number: 04-1116, MERCK Millipore Corporation, Billerica, MA, US) diluted with 3% nonfat milk in PBST. Membranes were washed three times in PBS Tween 0.1% and incubated with specific secondary antibodies diluted with 3% nonfat milk in PBST (goat anti-rabbit IgG (H + L)-HRP conjugate, diluted 1 : 10000, catalogue number: 172-1019, BioRad; goat anti-mouse IgG (H + L)-HRP conjugate, diluted 1 : 5000, catalogue number: 172-1011, BioRad) for 1 h at RT. The membranes were incubated with ECL reagents (BioRad) for 1 min and then were developed on Hyperfilm ECL (Amersham GE Healthcare, 28906835).
Images of the bands were digitized and the densitometry was performed using the open source image processing program ImageJ (http://imagej.nih.gov/ij/ ); β-actin bands were used for normalization.
Images of the bands were digitized and the densitometry was performed using the open source image processing program ImageJ (
3
CISD2-Beclin-1 Binding Evaluation
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A Pierce Co-IP kit (Thermo Fisher Scientific, Inc.) was used to examine the binding activity between CISD2 and beclin-1 in the indicated groups. The general procedure was performed according to the manufacturer's protocol, as previously described (25 (link)). The protein extracts were precipitated using anti-CISD2 (Sigma-Aldrich; Merck KGaA), and the precipitated protein was evaluated using western blot analysis with anti-beclin-1 (Sigma-Aldrich; Merck KGaA).
4
Evaluation of Autophagy in Spermatogonia Cells
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The mouse-derived spermatogonia cell line (GC-1 cell line) was bought from American Type Culture Collection (ATCC) and maintained in DMEM medium (Gibco, NY, USA) supplemented with 10% FBS (Gibco, NY, USA). Primary antibodies, anti-Beclin1 and anti-LC3, were from Sigma-Aldrich; anti-GAPDH was from Bioss Biotechnology. Secondary antibodies for immunoblot analysis were from Bioss Biotechnology. ALC was from Santa Cruz Biotechnology. Cell counting kit-8 (CCK-8) solution was from Dojindo Molecular Technologies Institute. Catalase (CAT) (A007-1-1), malondialdehyde (MDA) (A003-1-2), and total antioxidant capacity (T-AOC) (A015-1-2) assay kits were from Nanjing Jiancheng Bioengineering Institute (http://www.njjcbio.com/product.asp?cid=27&sort=saleorder ). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), monodansylcadaverine (MDC), 4′,6-diamidino-2-phenylindole (DAPI), and acridine orange/ethidium bromide (AO/EB) staining Kit were from Beyotime Biotechnology (https://www.beyotime.com/index.htm ). Trizol reagent and Prime Script reverse transcriptase reagent kit were from TaKaRa Biotechnology.
5
Lung Protein Extraction for Western Blot
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Lung tissue protein extraction was performed prior to Western blotting assay
conducted as described previously.15 (link) The primary Abs included rabbit anti-LC3B (Sigma-Aldrich, USA),
anti-Beclin1 (Sigma-Aldrich, USA), and anti-GAPDH (Servicebio, China). After
incubating with secondary Ab conjugated with HRP, immunoreactivity was developed
by an enterochromaffin-like system (Pierce).
conducted as described previously.15 (link) The primary Abs included rabbit anti-LC3B (Sigma-Aldrich, USA),
anti-Beclin1 (Sigma-Aldrich, USA), and anti-GAPDH (Servicebio, China). After
incubating with secondary Ab conjugated with HRP, immunoreactivity was developed
by an enterochromaffin-like system (Pierce).
6
Immunoblotting Analysis of Candida Infection
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The primary antibodies are as follows: anti-C. albicans (catalogue #GTX40096, Genetex), anti-LAMP1 (catalogue #1D4B, Developmental Studies Hybridoma Bank), anti-Alix (catalogue #sc-53540 Santa Cruz Biotechnology), anti-Tsg101 (catalogue #AB83 Abcam), anti-Galectin3 (catalogue #sc-32790 Santa Cruz Biotechnology), anti-LC3 (catalogue #PA1-16930 Thermo Fisher Scientific), anti-p62 (catalogue #51145 Cell Signalling), anti-Beclin1 (catalogue #A-00023 Sigma), and anti-Cx43 (catalogue #AB0016–500 SICGEN). Anti-goat secondary antibodies conjugated with Alexa Fluor 488 and horseradish peroxidase (HRP) were purchased from Invitrogen and Life Technologies Jackson, respectively. Anti-mouse secondary antibodies conjugated with Alexa Fluor 568, Alexa Fluor 647 and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rabbit secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 647, and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rat secondary antibodies conjugated with Alexa Fluor 594 and HRP were purchased from Invitrogen. Phalloidin was obtained from Sigma-Aldrich/Merck. LLOMe (L7393) and 3-MA (M9281-500MG) were purchased from Sigma-Aldrich/Merck. BAPTA-AM was obtained from Calbiochem (196419). Annexin V Alexa Fluor 488 Ready Flow Conjugate was purchased from Thermo Fisher Scientific (R37174).
7
Western Blot Analysis of Autophagy and Apoptosis
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The protein samples were prepared with RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor mix and boiled at 95°C for 10 min. The proteins were separated by 10% SDS gel and transferred to polyvinylidene fluoride (PVDF) membranes in a cold room. After that, the PVDF membranes were blocked with 5% skimmed milk [dissolved in Tris-buffered saline-Tween (TBST)] at room temperature (37°C) for 2 h and then incubated overnight with a primary antibody with gentle shaking at 4°C. After five washes with TBST, the membrane was incubated with a peroxidase-conjugated secondary antibody at room temperature (37°C) for 2 h. Following five washes with TBST, the target protein was visualized with chemiluminescence (ECL) (Thermo Fisher Scientific, Beijing, China). The primary antibodies used in this study include anti-LC3-I/II, anti-p62, anti-bECLin-1, and anti-Atg7 (Sigma-Aldrich, St. Louis, MO, USA) and anti-cleaved caspase 3 (Abcam, Cambridge, UK). GAPDH (Abcam) was used as an internal control.
8
Fatty Acid Autophagy Regulation in Hepatocytes
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Sodium palmitate and sodium oleate were obtained from Sigma‐Aldrich. Stock solution of 50 mM of each reagent was obtained by dissolution in H2O at 70°C. Working solutions were made by conjugation of 2 mM palmitic acid or free fatty acid (FFA) mixture (Sodium palmitate: sodium oleate = 1:2) with 2% BSA‐MEM/F‐12.36 Antibodies including anti‐LC3, anti‐Beclin‐1, anti‐ATG5 and anti‐β‐actin were obtained from Sigma‐Aldrich.
Mouse hepatocyte cell line AML‐12 was obtained from Shanghai Institutes for Biological Science, CAS. Cells were maintained in MEM/F12 medium supplemented with 10% foetal bovine serum, 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium and 40 ng/ml dexamethasone. Culture medium containing 0.5% lipoprotein depleted foetal bovine serum (LPDS) was used when FFA was added.
Mouse hepatocyte cell line AML‐12 was obtained from Shanghai Institutes for Biological Science, CAS. Cells were maintained in MEM/F12 medium supplemented with 10% foetal bovine serum, 10 μg/ml insulin, 5.5 μg/ml transferrin, 5 ng/ml selenium and 40 ng/ml dexamethasone. Culture medium containing 0.5% lipoprotein depleted foetal bovine serum (LPDS) was used when FFA was added.
9
Immunoblotting Analysis of Candida Infection
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The primary antibodies are as follows: anti-C. albicans (catalogue #GTX40096, Genetex), anti-LAMP1 (catalogue #1D4B, Developmental Studies Hybridoma Bank), anti-Alix (catalogue #sc-53540 Santa Cruz Biotechnology), anti-Tsg101 (catalogue #AB83 Abcam), anti-Galectin3 (catalogue #sc-32790 Santa Cruz Biotechnology), anti-LC3 (catalogue #PA1-16930 Thermo Fisher Scientific), anti-p62 (catalogue #51145 Cell Signalling), anti-Beclin1 (catalogue #A-00023 Sigma), and anti-Cx43 (catalogue #AB0016–500 SICGEN). Anti-goat secondary antibodies conjugated with Alexa Fluor 488 and horseradish peroxidase (HRP) were purchased from Invitrogen and Life Technologies Jackson, respectively. Anti-mouse secondary antibodies conjugated with Alexa Fluor 568, Alexa Fluor 647 and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rabbit secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 647, and HRP were purchased from Invitrogen and BioRad, respectively. Anti-rat secondary antibodies conjugated with Alexa Fluor 594 and HRP were purchased from Invitrogen. Phalloidin was obtained from Sigma-Aldrich/Merck. LLOMe (L7393) and 3-MA (M9281-500MG) were purchased from Sigma-Aldrich/Merck. BAPTA-AM was obtained from Calbiochem (196419). Annexin V Alexa Fluor 488 Ready Flow Conjugate was purchased from Thermo Fisher Scientific (R37174).
10
Immunohistochemical Analysis of Apoptosis Markers
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The expression of caspase-3, Light Chain 3 (LC3) and Beclin1 was measured by IHC staining. The samples were first washed in PBS (Ph 7.4) three times for 5 minutes each and boiled in 0.1% trisodium citrate for 15 minutes for antigen retrieval. Next, the sections were incubated with blocking reagent (3% milk and 5% fetal bovine serum; Absin Bioscience Inc., Shanghai, China) for 1 hour at room temperature and further incubated with anti-caspase-3 (1:500 dilution; Abcam, Cambridge, UK), anti-LC3 (1:200 dilution, Sigma, USA) and anti-Beclin1 (1:500 dilution, Sigma, St. Louis, MO, USA) antibodies at 4 °C overnight. Finally, after washing with PBS (pH 7.4), the tissues were exposed to a biotinylated anti-rabbit Immunoglobulin G (IgG) antibody (1:1000 dilution; Beyotime, Fuzhou, China) and a streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA). After the slides were sealed, the sections were imaged with a confocal microscope. The optical density was quantified using Image-Pro Plus version 7.0 (Media Cybernetics, Rockville, MD).
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