Anti becn1

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany

Anti-BECN1 is a laboratory antibody product that detects the BECN1 protein. BECN1 is a key regulator of autophagy, a cellular process involved in the recycling of damaged organelles and proteins. The Anti-BECN1 antibody can be used to identify and study the BECN1 protein in various experimental systems.

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BECN1 (19 citations) Anti-BECN1 antibody (2 citations)

8 protocols using anti becn1

Immunoprecipitation of Bcl-2, BECN-1, HIF-1α, and EZH2

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We extracted cell lysates from AGS and SNU-638 cells (2 × 10⁶/well) on a 100 mm cell culture plate in an IP buffer (pH 7.5) containing 50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, and protease inhibitor cocktail (Sigma). We incubated the antibodies anti-Bcl-2 (Santa Cruz), anti-BECN-1 (Santa Cruz), HIF-1α, and EZH2 (Cellsignaling) with lysate at 4 °C for 16 h. We used protein A/G Plus agarose (Santa Cruz) to pull down immunocomplexes. We washed precipitates three times with IP buffer. We resolved the immunoprecipitated proteins using 12% SDS-PAGE and analyzed them.

Kim T.W, & Lee H.G. (2021). Apigenin Induces Autophagy and Cell Death by Targeting EZH2 under Hypoxia Conditions in Gastric Cancer Cells. International Journal of Molecular Sciences, 22(24), 13455.

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Vitamin D Analog TX 527 Protocol

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Vitamin D analog TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)₂D₃] was originally synthesized by M. Vandewalle and P. De Clercq (University of Ghent, Ghent, Belgium) and provided by Théramex (Monaco). Immobilon P (polyvinylidenedifluoride; PVDF) membranes, 1α,25(OH)₂D₃, the antibiotic G418 and LY294002 were from Sigma-Aldrich (St. Louis, MO, USA). Puromycin supplied by Invivogen (San Diego, CA, USA). The antibodies used were rabbit monoclonal anti-p-Akt, anti-Akt, anti-MEKα (Cell Signaling Technology, Danvers, MA, USA) and anti-Tubulin (Thermo Fisher Scientific Inc., Waltham, MA, USA); mouse monoclonal anti-LC3, anti-BECN1, anti-p-mTOR, and anti-mTOR, goat polyclonal Lamin B and secondary antibodies anti-rabbit, anti-mouse and anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Roche Applied Science supplied high Pure RNA Isolation Kit (Indianapolis, IN, USA). High Capacity cDNA RT and SYBR Green PCR Master Mix reagent (Applied Biosystems) were acquired from Thermofisher (Thermofisher, Buenos Aires, AR). GAPDH primer was from Invitrogen^TM (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA, USA) and BECN1 primer was obtained by Eurogentec (Serain, Belgium). The inhibitor of flux autophagy, Chloroquine, was kindly provided by Dr. Daniel Grasso (IBIMOL Universidad de Buenos Aires – CONICET).

Suares A., Tapia C, & González-Pardo V. (2019). VDR agonists down regulate PI3K/Akt/mTOR axis and trigger autophagy in Kaposi's sarcoma cells. Heliyon, 5(8), e02367.

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Immunohistochemical Evaluation of Autophagy Markers

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Lymph nodes were embedded in paraffin and sections were deparaffinized, rehydrated, and subjected to high temperature antigen retrieval in 10 mM sodium citrate buffer pH 6,0. Immunohistochemistry was performed as previously reported,⁵² (link) in tissue sections and fixed PBMC. The primary antibodies utilized were: rabbit polyclonal anti-AMBRA1 (1:100, ProSci, 4557) rabbit polyclonal anti-BECN1 (1:50, Santa Cruz Biotechnology, sc-11427), rabbit polyclonal anti-ATG5 (1:50, Santa Cruz Biotechnology, sc-33210).
The percentage of positive PBMC/total PBMC, was counted for AMBRA1, BECN1, and ATG5 stainings. Three independent observers evaluated the number of positive cells by using a light microscope without the knowledge of clinical diagnosis. A minimum of 500 PBMC/patient were analyzed. The percentage of positive lymph node cells/total cells, was counted for AMBRA1, BECN1 and ATG5 stainings. For each slide, a minimum of 10 fields was examined at 40× magnification.

Nardacci R., Amendola A., Ciccosanti F., Corazzari M., Esposito V., Vlassi C., Taibi C., Fimia G.M., Del Nonno F., Ippolito G., D’Offizi G, & Piacentini M. (2014). Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients. Autophagy, 10(7), 1167-1178.

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Nanoparticle Uptake and Autophagy Imaging

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Cells were cultured on cover slides and treated with 150 µg of nanoparticles for indicated time periods, then fixed in ice-cold 4% paraformaldehyde/PBS. For colocalization experiments, cells were transfected in advance with GFP-Rab5orGFP-Rab9 plasmids and treated with nanoparticles after 48 h. Cells were fixed in ice-cold 4% paraformaldehyde/PBS. Following the fixation, nuclei were stained using Hoechst (Invitrogen, 31716 W) in PBS. Coverslides were mounted onto glass slides, and samples were analyzed with a Carl Zeiss LSM 710 confocal microscope (Zeiss, Germany). For indirect immunostaining experiments, following fixation, cells were permeabilized in PBS containing 0.1% BSA (Sigma, #A4503) and 0.1% saponin (Sigma, #84510). As primary antibodies, anti-LC3 (Sigma, L8918), anti-BECN1 (Santa Cruz, sc-11427) and anti-ATG4C (Sigma-Aldrich, AB75056) were used. Anti-mouse Alexa Fluor 488 (Invitrogen, #A32723) and anti-rabbit (Invitrogen, #A-11008) were used as secondary antibodies. Coverslides were mounted onto glass slides, and samples were analyzed using a BX60 fluorescence microscope (Olympus, BX60) or Carl Zeiss LSM 710 confocal microscope (Zeiss, Germany).

Unal O., Akkoc Y., Kocak M., Nalbat E., Dogan-Ekici A.I., Yagci Acar H, & Gozuacik D. (2020). Treatment of breast cancer with autophagy inhibitory microRNAs carried by AGO2-conjugated nanoparticles. Journal of Nanobiotechnology, 18, 65.

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Autophagy Regulation by P2RX7 Signaling

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The following antibodies were used: anti-P2RX7 (Synaptic Systems, 177003) 1:1000, anti-MAPK1-MAPK3 (Cell Signaling Technology, 9102) 1:2000, anti-phospho-MAPK1-MAPK3 (Cell Signaling Technology, 9106) 1:1000, anti-ACTB (Sigma, A2066) 1:1000, anti-LC3B (Sigma, L7543) 1:1000, anti-GAPDH (Sigma, G9545) 1:1000, anti-COX4/COXIV (Abcam, ab14744) 1:000, anti-BECN1 (Santa Cruz Biotechnology, 48381) 1:200. Other chemicals used were as follows: P2RX7 antagonists A438079 and A804598 (Tocris Bioscience, 2972 and 4473, respectively) and Brilliant Blue G (BBG, Ascent Scientific, ASC-389), HSPA2 and HSP90 inhibitors, VER155008, geldanamycin and 17-DMAG (Tocris Biosciences, 3803, 1368 and 2610, respectively), Proteostat aggresome detection kit (Enzo Life Sciences, ENZ-51035-K100), HSPA2 inhibitor KNK437 (Merck Millipore, 373260), the cell permeable, fluorogenic CASP3-CASP7 substrate DEVD-Nucview488 (Cambridge Biosciences, BT10403) and the mitochondrial membrane potential-sensitive probe tetramethylrhodamine ethyl ester (TMRE; Sigma, 87917), digoxin and digitoxigenin (Sigma, D6770 and D9404, respectively).

Young C.N., Sinadinos A., Lefebvre A., Chan P., Arkle S., Vaudry D, & Gorecki D.C. (2015). A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90. Autophagy, 11(1), 113-130.

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Western Blot Analysis of Autophagy Proteins

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Proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred on either polyvinylidene fluoride membranes or nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). Membranes were probed using the following antibodies: anti-p62 (Abcam, Cambridge, UK); anti-Atg5, anti-P-Atg14 (Ser29), anti-Beclin-1, anti-FLIP, anti-LC3, anti-NEDD4, anti-mTOR, anti-P-mTOR (Ser2448), anti-ULK1, anti-P-ULK1 (Ser757), anti-V5, anti-Vps34 (Cell Signaling Technology); anti-BECN1, anti-GFP, anti-HA (Santa Cruz Biotechnology, Heidelberg, Germany); and anti-β-Actin, anti-Tubulin (Sigma-Aldrich). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Bio-rad, Hercules, CA, USA). Membranes were washed with Tris-buffered saline (Medicago, Danmarks-Berga, Uppsala, Sweden) with 0.1% Tween-20 (Sigma-Aldrich) and developed through the chemiluminescence system (Amersham Bioscience) on the ChemiDock image analyser (Bio-Rad), which was also used for densitometric quantifications.

Tomaipitinca L., Petrungaro S., D’Acunzo P., Facchiano A., Dubey A., Rizza S., Giulitti F., Gaudio E., Filippini A., Ziparo E., Cecconi F, & Giampietri C. (2021). c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability. Cell Death & Disease, 12(7), 686.

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Immunoprecipitation of Bcl-2 and Beclin-1

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We extracted cell lysates from AGS cells (2 × 10⁶ per well) on 100 mm cell culture plate in a IP buffer (pH 7.5) containing 50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 0.5%(v/v) NP-40, and protease inhibitor cocktail (Sigma). We incubated anti-Bcl-2 (Santa Cruz) and anti-BECN-1 (Santa Cruz) with lysate at 4 °C for 16 h. We used protein A/G PLUS agarose (Santa Cruz) to pull down immunocomplexes. We washed precipitates three times with IP buffer. We resolved the immunoprecipitated proteins by 12% SDS-PAGE and analyzed them.

Kim T.W., Lee S.Y., Kim M., Cheon C, & Ko S.G. (2018). Kaempferol induces autophagic cell death via IRE1-JNK-CHOP pathway and inhibition of G9a in gastric cancer cells. Cell Death & Disease, 9(9), 875.

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Immunoblotting Analysis of Cellular Signaling

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Cells treated as described, were processed as previously indicated [21 (link)] and probed with anti-MAP- LC3 (H-50) rabbit polyclonal antibody (sc-28226, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), anti-p53 (DO 1) mouse monoclonal antibody (sc-126, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), anti-p21 (C-19) rabbit polyclonal antibody (sc-397, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), anti-SGK1 rabbit polyclonal antibody (#07-315, MERCK MILLIPORE), anti-BIP(GRP-78) (C50B12) rabbit polyclonal antibody (#3177, Cell Signaling Technology, Inc.) anti-GAPDH (sc-25778, Santa Cruz Biotechnology, Inc. Santa Cruz, CA), Anti BECN1 (sc-48381, Santa Cruz Biotechnology, Inc. Santa Cruz, CA)

Talarico C., Dattilo V., D'Antona L., Barone A., Amodio N., Belviso S., Musumeci F., Abbruzzese C., Bianco C., Trapasso F., Schenone S., Alcaro S., Ortuso F., Florio T., Paggi M.G., Perrotti N, & Amato R. (2016). SI113, a SGK1 inhibitor, potentiates the effects of radiotherapy, modulates the response to oxidative stress and induces cytotoxic autophagy in human glioblastoma multiforme cells. Oncotarget, 7(13), 15868-15884.

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