Cell lysates collected in RIPA buffer (supplemented with 1 mM PMSF, 1× protease inhibitor) were separated by SDS–PAGE, transferred to a PVDF membrane (Immobilon-P, Merck Millipore Ltd), and incubated with primary antibodies (1:1000) at 4 °C overnight. After subsequent staining with HRP-conjugated secondary antibodies (1:10000 for anti-rabbit and 1:3000 for anti-mouse antibodies; 4 °C; overnight), the signal was developed using the ECL Star kit (Euroclone SpA, according to the instruction of the manufacturer using the BioRad ChemidocTM imaging system (Image LabTM touch software, BioRad). β2-actin was used as the loading control. Western blot experiments were performed with anti-ATG5, anti-PU.1, anti-Beclin1, anti-IRF1 (D5F5U; 9G7; D40C5; D5E4, Cell Signaling Technology), anti-LC3I/LCRII (MBL-PM036, MBL International), anti-CRLS1 (PA5–25338, Invitrogen), anti-LPCAT1, anti-PGS1 (PA5–26318; PA5–43247, Invitrogen) and anti-β2 actin (SC-47778, Santa Cruz) antibodies.
Anti beclin 1
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China, Germany
Anti-Beclin-1 is a primary antibody that recognizes the Beclin-1 protein. Beclin-1 is a key regulator of autophagy, a cellular process involved in the degradation and recycling of damaged or unwanted cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3, by Cell Signaling Technology (42 mentions)
Anti p62, by Cell Signaling Technology (40 mentions)
Anti lc3b, by Cell Signaling Technology (36 mentions)
Anti atg5, by Cell Signaling Technology (32 mentions)
Anti gapdh, by Cell Signaling Technology (25 mentions)
Anti β actin, by Cell Signaling Technology (25 mentions)
Anti atg7, by Cell Signaling Technology (24 mentions)
Anti caspase 3, by Cell Signaling Technology (23 mentions)
Anti bax, by Cell Signaling Technology (21 mentions)
Anti lc3a b, by Cell Signaling Technology (19 mentions)
Anti akt, by Cell Signaling Technology (17 mentions)
Anti mtor, by Cell Signaling Technology (16 mentions)
Anti bcl 2, by Cell Signaling Technology (16 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (16 mentions)
Anti p mtor, by Cell Signaling Technology (13 mentions)
Pvdf membrane, by Merck Group (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Anti p akt, by Cell Signaling Technology (11 mentions)
Anti ampk, by Cell Signaling Technology (11 mentions)
Anti parp, by Cell Signaling Technology (11 mentions)
186 protocols using anti beclin 1
1
Western Blot Analysis of Autophagy Proteins
2
Western Blot Analysis of Autophagy Markers
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Protein extracts from cultured cells were obtained by homogenization in RIPA buffer (Santa Cruz Biotechnology), then cleared by centrifugation at 12,000 rpm for 20 min. For immunodetection, 50 µg total protein from tumor tissue or cultured cells were mixed with Laemmli sample buffer, boiled, separated in 12 or 15% SDS-PAGE, and transferred in a PVDF membrane (Amersham-GE Healthcare). Membranes were incubated overnight using a 1:1,000 (v/v) dilution of the anti-ULK1 (Cell Signaling), 1:3,000 of the anti-Beclin-1 (Cell Signaling), 1:3,000 of the anti-LC3 (Cell Signaling), 1:3,000 of the anti-ATG4b (Santa Cruz), and 1:3,000 of the anti-HIF-1alpha (GeneTex); for detection, 1:2,500 (v/v) dilutions of HRP anti-rabbit or anti-mouse conjugate antibodies (Santa Cruz Biotechnology) were used. Finally, using the Super Signal West Femto chemiluminescent substrate (Thermo Scientific), the membranes were scanned in the C-Digit blot scanner (Li-Cor) and the images were analyzed for densitometry in the associated Image Studio software (LiCor). Membranes were stripped and re-probed for detection of actin (anti-actin, Sc-47778) as a loading control. A representative image from three independent experiments is shown.
3
MTT and DMSO Assay for Cell Viability
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI medium, Dulbecco’s minimum essential medium (DMEM), fetal bovine serum, horse serum, and Fura-2 AM were obtained from Invitrogen (Carlsbad, CA). Anti-LC3, anti-p62, anti-beclin-1, anti-PI3K, anti-phosphorylated (p)-PI3K, anti-Akt, anti-p-Akt, anti-mTOR, anti-p-mTOR, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). N-Adamantyl-4-methylthiazol-2-amine (KHG26693) was synthesized and characterized as previously described (16 (link)).
4
Protein Extraction and Western Blot Analysis
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Cells were plated and treated as described in section 2.3 . After 24 h, culture medium was removed, and cells were washed with PBS. To obtain total protein extracts, cell lysis was performed chemically, with Lysis Buffer (5 mM Hepes pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1.5 mM EGTA, 4 nM PMSF, 1% Aprotinine, 20 nM sodium pyrophosphate, 10 nM sodium orthovanadate), and mechanically, with a cell scarper.
Total protein samples were then clarified by centrifugation (13,000g, 15 min, 4 °C) and quantified using Bradford method.
10 μg of proteins were loaded in a polyacrylamide gel and after SDS-PAGE electrophoresis, western blotting was performed as described by antibodies manufacturers (anti-ERα, 1:1000, Santa Cruz, USA; anti-BRCA1, 1:50, Cell Signaling, USA; anti-CAV1, 1:1000, Cell Signaling, USA; anti-actin, 1:1000, Santa Cruz, USA; anti-LC3, 1:1000, Cell Signaling, USA; anti-Beclin1, 1:1000, Cell Signaling, USA; anti-rabbit, 1:2000, PerkinElmer, USA; anti-mouse, 1:2000, Chemicon, USA; anti-goat, 1:2000, Chemicon, USA).
Total protein samples were then clarified by centrifugation (13,000g, 15 min, 4 °C) and quantified using Bradford method.
10 μg of proteins were loaded in a polyacrylamide gel and after SDS-PAGE electrophoresis, western blotting was performed as described by antibodies manufacturers (anti-ERα, 1:1000, Santa Cruz, USA; anti-BRCA1, 1:50, Cell Signaling, USA; anti-CAV1, 1:1000, Cell Signaling, USA; anti-actin, 1:1000, Santa Cruz, USA; anti-LC3, 1:1000, Cell Signaling, USA; anti-Beclin1, 1:1000, Cell Signaling, USA; anti-rabbit, 1:2000, PerkinElmer, USA; anti-mouse, 1:2000, Chemicon, USA; anti-goat, 1:2000, Chemicon, USA).
5
Western Blot Analysis of Autophagy Markers
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Mouse tissue extracts were prepared by homogenizing tissues in lysis buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, proteinase inhibitor cocktail (Roche Applied Sciences, Mannheim, Germany), and Halt phosphatase inhibitor cocktail (Roche Applied Sciences), and then subjected to Western blot analysis with anti-LC3 (1:500, Sigma-Aldrich), anti-P62 (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-Bnip3 (1:500, Cell Signaling Technology), anti-AMPK (1:500, Cell Signaling Technology), anti-p-AMPK (1:500, Cell Signaling Technology), anti-Beclin1 (1:500, Cell Signaling Technology), and anti-GAPDH (1:3,000, Cell Signaling Technology) antibodies. Activation of AMPK was expressed as the ratio of phosphorylated AMPK (pAMPK) to total AMPK (tAMPK).
6
Comprehensive Western Blot Antibody Panel
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In this work we used the following primary antibodies: mouse monoclonal anti-STAT3 (1:1000) (BD Transduction Laboratories, 610189), mouse monoclonal anti-phosphoSTAT3 (1:100) (pY705) (BD Transduction Laboratories, 612356), rabbit polyclonal anti-PARP p85 Fragment pAb (1:500) (Promega, G7341), rabbit polyclonal anti-Mcl-1 (1:500) (Cell Signaling, 5453P) and goat polyclonal anti-caspase 3 (Santa Cruz Biotechnology Inc., sc-1225). To study autophagy we used the following primary antibodies: rabbit polyclonal anti-LC3 (1:1000) (Novus Biologicals, NB100-2220SS), mouse monoclonal anti-p62 (1:1000) (BD Transduction Laboratories, 610832) and rabbit polyclonal anti-Beclin1 (1:1000) (Cell Signaling, 3738S).
Monoclonal mouse anti-β-actin (1:10000) or anti-GAPDH (1:1000) (Santa Cruz Biotechnology Inc., sc-137179) were used as markers of equal loading” in “were used as markers of equal loading. Goat polyclonal anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., sc-2005) rabbit polyclonal anti-goat IgG-HRP (Santa Cruz Biotechnology Inc., sc-2768) and rabbit IgG-HRP (Santa Cruz Biotechnology Inc., sc-2004) were used as secondary antibodies. All the primary and secondary antibodies used in this study were diluted in a PBS- 0.1% Tween 20 solution containing 3% BSA.
Monoclonal mouse anti-β-actin (1:10000) or anti-GAPDH (1:1000) (Santa Cruz Biotechnology Inc., sc-137179) were used as markers of equal loading” in “were used as markers of equal loading. Goat polyclonal anti-mouse IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., sc-2005) rabbit polyclonal anti-goat IgG-HRP (Santa Cruz Biotechnology Inc., sc-2768) and rabbit IgG-HRP (Santa Cruz Biotechnology Inc., sc-2004) were used as secondary antibodies. All the primary and secondary antibodies used in this study were diluted in a PBS- 0.1% Tween 20 solution containing 3% BSA.
7
Autophagy and Apoptosis Signaling Pathway
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Trypan blue solution (prod. no. T8154), phosphatase inhibitor cocktails 2 and 3 (prod. no. P5726 and P0044, respectively), and furazolidone (prod. no. V900742) were from Sigma-Aldrich (St. Louis, MO, USA). A protease inhibitor cocktail (ref. no. 11836 153 001) was from Roche Diagnostics (Basel, Switzerland). Primary antibodies anti-Atg5 (#2630), anti-beclin-1 (#3738), cleaved caspase-3 (#9661), Bcl-2 (#3498S), PARP (#46D11), H2AX (#2595S), LC3I/II (#4180S), BAX (#2772), and P53 (#9282) were purchased from Cell Signaling Technology (Danvers, MA USA), and anti-actin was purchased from Sigma-Aldrich (prod. no. A3853). Secondary antibodies goat anti-rabbit IgG- (H+L) HRP conjugate (cat. no. 170-6515) and goat anti-mouse IgG- (H+L) HRP conjugate (cat. no. 170-6516) were obtained from Bio-Rad Laboratories (Mississauga, Ontario, Canada).
8
Protein Isolation and Western Blot Analysis
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Total protein was isolated from lung tissues and RAW264.7 cells using RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was measured using a BCA kit (KeyGen Biotech). An equal volume per sample was subjected to 10% SDS-PAGE and the proteins were transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were blocked in 5% non-fat milk and probed with anti-GGPPS1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-IL-1β (1:1000, Abcam), anti-IL-6 (1:1000, Abcam), anti-IL-18 (1:200, Abcam), anti-TNF-α (1:1000, Abcam), anti-LC3B (1:200, Abcam), anti-ATG5 (1:1000, Cell Signaling Technology, Danvers, MA), anti-Beclin1 (1:1000, Cell Signaling Technology), anti-Rab37 (1:500, Abcam), and anti-Actin (1:1000, Santa Cruz Biotechnology). After washing, the membranes were probed with HRP-conjugated secondary antibodies (1:2000, Abcam) and the signal was detected by the classical ECL method.
9
Western Blot Analysis of Cellular Stress Markers
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Protein extraction and western blot analysis were performed as previously described [52 (link)]. Anti-Beclin-1, anti-cleaved caspase 7, anti-CHOP, anti-GRP78, anti-phospho-H2AX, anti-LC3, anti-PARP, and anti-p62 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-GRP94 was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Anti-α-tubulin was obtained from Abcam (Eugene, OR, USA).
10
Protein Expression Analysis in Rat Brain
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Protein was extracted from the rat brain or the cultured cells with RIPA lysis buffer (Sigma-Aldrich) on ice. The supernatants were collected after centrifugation at 12000×g at 4°C for 20min. Protein concentration was determined using a BCA protein assay kit (Bio-rad, China), and the proteins were separated on SDS-polyacrylamide gels, and then transferred to a PVDF membrane. The membrane blots were first probed with a primary antibody. After incubation with horseradish peroxidase-conjugated second antibody, autoradiograms were prepared using the enhanced chemiluminescent system to visualize the protein antigen. The signals were recorded using X-ray film. Primary antibodies were rabbit anti-caspase 3, anti-Beclin-1, anti-LC3, anti-BNIP3, anti-NIX and anti-β-actin (Cell Signaling, San Jose, CA, USA). Secondary antibody is HRP-conjugated anti-rabbit (Jackson ImmunoResearch Labs, West Grove, PA, USA). β-actin was used as a protein loading control. The protein levels were first normalized to β-actin, and then normalized to the experimental controls. HIF-1a levels were determined by an ELISA kit (R&D System, Los Angeles, CA, USA).
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