Becn1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, China

BECN1 is a gene that encodes the Beclin 1 protein, a key regulator of autophagy, a cellular process that involves the degradation and recycling of damaged or unwanted cellular components. The Beclin 1 protein is involved in the initiation and regulation of the autophagy process.

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Lab products found in correlation

34 protocols using becn1

Autophagy-related Protein Detection

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Stock solutions of Bafilomycin A1 (LC Laboratories, B-1080), MG132 (Calbiochem, 474790), Cycloheximide (Sigma, 01810) and Rapamycin (Enzo, BML-A275-0025) were prepared in DMSO (Roth, A994.2). Stock solution of Canavanine (Santa Cruz Biotech, sc-202983A) and Puromycin (Sigma, P8833) was prepared in distilled H2O.
Antibody sources were as follows: for Actin (Sigma, A5060), BAG1 (Abcam, ab7976), BAG3 (Proteintech Group, 10599-1-AP), BECN1 (Cell Signaling, 3495), CTSD (Abcam, ab75852), DLP1 (BD Transduction Laboratories, 611113), LAMP2 (DSHB Biology, ABL-93), LC3B (Nanotools, 0260-100), LC3B (Sigma, L7543), OPA1 (BD Transduction Laboratories, 612607), Phospho mTOR (Abcam, ab109268), Puromycin (Millipore, MABE343), mTOR (Calbiochem, OP97), p62 (Progen, GP62-C), PIK3C3 (Cell Signaling, 4263), Poly-Ubiquitin (Dako, Z0458), RAB18 (Sigma, SAB4200173), Tubulin (Millipore, MAB1637), Tubulin (Sigma, T9026), TFEB (Proteintech Group, 13372-1-AP), Vimentin (SCBT, sc-373717), WIPI1 (Sigma, HPA007493).

Chakraborty D., Felzen V., Hiebel C., Stürner E., Perumal N., Manicam C., Sehn E., Grus F., Wolfrum U, & Behl C. (2019). Enhanced autophagic-lysosomal activity and increased BAG3-mediated selective macroautophagy as adaptive response of neuronal cells to chronic oxidative stress. Redox Biology, 24, 101181.

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Autophagic Flux Measurement in Oral Cancer Cells

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Parental or shRNA Ca9-22 and OECM-1 cells were treated with or without 20 μM CQ for 1 h prior to LPLI treatment. The recovered LPLI-treated cell lysates were used to detect the accumulation of MAP1LC3-II, a lipidated and membrane-bound form of MAP1LC3, by immunoblotting to determine autophagic flux [21 (link)]. For immunoblotting, the cells were briefly rinsed with PBS (Biological Industries, Kibbutz Beit-Haemek, Israel) and lysed with an lysis buffer [1% Triton X100, 50 mM Tris HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA and a protease inhibitor cocktail (Roche Life Science)]. The proteins in the cell lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies against MAP1LC3 and ACTB (β-actin) (Sigma-Aldrich), SQSTM1 (BD Pharmingen), ATG7, RelA S536-P, and BECN1 (Cell Signaling Technology) overnight at 4°C. The proteins were probed with an HRP-labeled secondary antibody (Santa Cruz Biotechnology) and detected using an ECL reagent. The membranes were scanned and analyzed for protein expression level using a ChemiDoc XRS Imaging System (Bio-Rad Laboratories).

Shu C.W., Chang H.T., Wu C.S., Chen C.H., Wu S., Chang H.W., Kuo S.Y., Fu E., Liu P.F, & Hsieh Y.D. (2016). RelA-Mediated BECN1 Expression Is Required for Reactive Oxygen Species-Induced Autophagy in Oral Cancer Cells Exposed to Low-Power Laser Irradiation. PLoS ONE, 11(9), e0160586.

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Autophagy-related Protein Detection

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ATG2A (#15011), ATG5 (#2630), ATG7 (#2631), BECN1 (#3738), BIRC2 (D5G9), CASP8 (1C12), FADD (#2782), FAS (C18C12), FASLG (#4273), HSP90 (C45G5); LAMP1 (C54H11), MLKL (D2I6N), PARP1 (46D11), RIPK1 (D94C12), RIPK3 (D4G2A), ULK1 (D9D7), and XIAP (3B6) antibodies were obtained from Cell Signaling. SQSTM1 (#ab56416) and STX17 (#ab116113) antibodies were purchased from Abcam. β-actin (ACTB; AC-74) and ATG2B (#HPA019665) were purchased from Sigma. MAP1LC3B (#NB100-2220) was purchased from Novus Biologicals. Cell lysis, co-immunoprecipitation, subcellular fractionation, and western blotting were performed as previously described^{19 (link),41 (link)}. Relative densities of the target bands to the reference bands was calculated using ImageJ (NIH).

Campbell G.R., To R.K., Zhang G, & Spector S.A. (2020). SMAC mimetics induce autophagy-dependent apoptosis of HIV-1-infected macrophages. Cell Death & Disease, 11(7), 590.

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Multi-Omics Evaluation of Anticancer Compounds

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Vitexin, acridine orange (AO), Tricaine, 5-fluorouracil (5-FU), cisplatin (CIS), docetaxel (DOC), vincristine (VIN), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Hoechst 33342, 3-methyladenine (3-MA), dimethylsulfoxide (DMSO), dichloro-dihydrofluorescein diacetate (DCFH-DA) and other chemicals, unless stated otherwise, were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-actin, MDR-1, HSF-1, lamin B, HSP70, HSP27, BID, BAX, cytochrome C, caspase-3, caspase-9, and Bcl-2 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HSP90 antibody was purchased from Enzo Life Science (Farmingdale, NY, USA). BECN-1 and ATG5 were purchased from Cell Signaling Technology (Beverly, MA, USA). LC3 was obtained from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies for western blot analysis were purchased from Santa Cruz Biotechnology.

Bhardwaj M., Cho H.J., Paul S., Jakhar R., Khan I., Lee S.J., Kim B.Y., Krishnan M., Khaket T.P., Lee H.G, & Kang S.C. (2017). Vitexin induces apoptosis by suppressing autophagy in multi-drug resistant colorectal cancer cells. Oncotarget, 9(3), 3278-3291.

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Western Blot Analysis of Autophagy Proteins

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Cells were collected and lysed in 1 × RIPA buffer (Sigma, MO, USA) and then subjected to Western blot as described previously^{46 (link)}. 80 µg of each protein extract was separated by 6~15% SDS-PAGE gels and transferred to nitrocellulose membrane. The immunoreactive bands were firstly incubated with the primary antibodies, including HMGN5 (1:500), ATG5 (1:1000), BECN-1 (1:2000), and β-actin (1:2000) (Cell Signaling Technology, MA, USA). Then, the immunoreactive bands were incubated with the secondary antibody and visualized using ECL-PLUS Kit (Beyotime Institution of Biotechnology, Haimen, China).

Meng Y., Gao R., Ma J., Zhao J., Xu E., Wang C, & Zhou X. (2017). MicroRNA-140-5p regulates osteosarcoma chemoresistance by targeting HMGN5 and autophagy. Scientific Reports, 7, 416.

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Cell Apoptosis Signaling Protocol

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Phosphate-buffered saline, dimethyl sulfoxide, Dulbecco modified Eagle’s medium, fetal calf serum, L-glutamine, penicillin, and streptomycin were obtained from Sigma-Aldrich (St Louis, MO, USA). Antibodies against caspase-3, Bcl-2, becn-1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). All other reagents were of the highest purity among commercially available products.

Han A.R., Yang J.W., Na J.M., Choi S.Y, & Cho S.W. (2019). Protective effects of N,4,5-trimethylthiazol-2-amine hydrochloride on hypoxia-induced β-amyloid production in SH-SY5Y cells. BMB Reports, 52(7), 439-444.

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Investigating Autophagy Signaling Proteins

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The antibodies against S100A8 and p62 were obtained from Santa Cruz Biotechnology (Sana Cruz, CA, USA). The antibodies to Actin, BECN1, PI3KC3, C-PARP, ULK1, Bcl-2 and P-ULK1 were from Cell Signaling Technology (Boston, MA, USA). The antibodies to LC3 and TLR-4 were purchased from Abcam (Cambridge, MA, USA). Anti-Atg7 antibody was from Novus (Denver-Littleton, CO, USA). Vincristine (VCR), adriamycin (ADM), rotenone (Rot), thenoyltrifluoroacetone (TTFA), antimycin A (AA), E64D, anti-RAGE antibody and pepstatin were from Sigma (Milpitas, CA, USA). Full-length human S100A8 cDNA (pLPCX-S100A8) was a gift from Dr. RW Stam (Erasmus Medical Center/Sophia Children's Hospital, Netherlands). FITC-Annexin V Apoptosis Detection kit and the Nuclear and Cytoplasmic Protein Extraction kit were purchased form Beyotime Institute of Biotechnology (Beijing, China). S100A8 protein was obtained from Novus Biologicals. Contaminating LPS was removed by Triton X-114 extraction. LPS content was always below 0.5 ng/mg protein, which did not cause an effect in our assays.

Yang M., Zeng P., Kang R., Yu Y., Yang L., Tang D, & Cao L. (2014). S100A8 Contributes to Drug Resistance by Promoting Autophagy in Leukemia Cells. PLoS ONE, 9(5), e97242.

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Cryptotanshinone Signaling Pathway Analysis

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CQ (Sigma, C6628) was dissolved in phosphate buffered saline (PBS, Sigma, 08057), and NH₄Cl (Sigma, 09718) was dissolved in sterile H₂O. Cryptotanshinone (CPT) was obtained from MedChemExpress (MCE, HY-N0174). ATG7, GAPDH, STAT3, and JAK2 were obtained from Santa Cruz Biotechnology (sc-376212, sc-47724, sc-8019, and sc-390539). E-cadherin, vimentin, BECN1, STAT3, and p-STAT3 were purchased from Cell Signaling Technology (3195, 5741, 3738, 4904, and 9145).

Hu F., Li G., Huang C., Hou Z., Yang X., Luo X., Feng Y., Wang G., Hu J, & Cao Z. (2020). The autophagy-independent role of BECN1 in colorectal cancer metastasis through regulating STAT3 signaling pathway activation. Cell Death & Disease, 11(5), 304.

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Protein expression analysis in cells

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Western blot, immunohistochemistry (IHC) and immunofluorescence (IF) assays were performed as previously described^{22 (link)}. Antibodies for PRAS40, BECN1, LC3B, Ki-67, SQSTM1, mTOR, LAMP1, GAPDH and β-tubulin were all purchased from Cell Signaling Technology (CST), and the expression of GAPDH and β-tubulin were used as internal controls.

Xie Y., Chen L., Zhou J., Yang C., Xu C., Fan X., Tan Y., Wang Y., Kang C, & Fang C. (2020). TGFβ signaling-induced miRNA participates in autophagic regulation by targeting PRAS40 in mesenchymal subtype of glioblastoma. Cancer Biology & Medicine, 17(3), 664-675.

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Protein Expression Analysis in Cells

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Cells or exosomes were harvested and lysed in RIPA buffer. Protein concentrations were detected with a BCA assay kit (P0012S, Beyotime, Shanghai, China). Transferred membranes were incubated overnight at 4 °C with the following primary antibodies from Cell Signaling Technology: MAP 1LC3B (2775), BECN1 (3495), ATG5 (2630), ATG7 (2631), AMP-activated protein kinase (AMPK, 5831), phosphorylated-(p-)AMPK (Thr172, 2535), and β-actin (3700). Additional antibodies that were used were as follows: GCK (Abcam, ab155962), PFK (Abcam, ab204131), PK (Abcam, ab171744), p-GSK3β (Abcam, ab68476), GSK3β (Abcam, ab62368), G-6-P (Abcam, ab167394), PEPCK (Abcam, ab133603), PPARα (Abcam, ab8934), and SREBP-1c (Proteintech, 14088-1-AP). After incubation with horseradish peroxidase-labeled secondary antibodies, protein bands were exported by the Image Lab software (BioRad, USA). Protein band intensities were measured via ImageJ and were normalized to β-actin.

He Q., Wang L., Zhao R., Yan F., Sha S., Cui C., Song J., Hu H., Guo X., Yang M., Cui Y., Sun Y., Sun Z., Liu F., Dong M., Hou X, & Chen L. (2020). Mesenchymal stem cell-derived exosomes exert ameliorative effects in type 2 diabetes by improving hepatic glucose and lipid metabolism via enhancing autophagy. Stem Cell Research & Therapy, 11, 223.

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