Anti lc3b 1 2

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-LC3B-I/II is a primary antibody product used to detect the LC3B protein, which is involved in the autophagy process. The antibody recognizes both the cytosolic (LC3B-I) and lipidated (LC3B-II) forms of the protein.

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9 protocols using anti lc3b 1 2

Western Blot Analysis of Cellular Signaling

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Cell lysates were standardized for protein content, resolved on 4% to 12% SDS polyacrylamide gels, and blotted onto nitrocellulose membranes. Membranes were probed with rabbit anti-pPKM2, anti-PKM2, anti-CHOP, anti-GRP78/BIP, anti-pEGFR (Y1086), anti-EGFR, anti-LC3B-I/II, and anti-β-actin (Cell Signaling). Antibody binding was detected by using an ECL Chemiluminescence Kit (Thermo Scientific).

Sobhakumari A., Orcutt K.P., Love-Homan L., Kowalski C.E., Parsons A.D., Knudson C.M, & Simons A.L. (2016). 2-Deoxy-d-glucose Suppresses the In Vivo Antitumor Efficacy of Erlotinib in Head and Neck Squamous Cell Carcinoma Cells. Oncology Research, 24(1), 55-64.

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Protein Extraction and Western Blot Analysis

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Total protein was isolated from the primary cardiomyocytes using RIPA buffer (Beyotime Biotechnology, Shanghai, China) with the protease inhibitor cocktail. Protein concentration was quantified with BCA protein assays kit (Beyotime Biotechnology, Shanghai, China). 30-70 μg of proteins was separated by SDS-PAGE gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked by 5% nonfat dry milk with PBS containing 0.1% Tween-20 at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase- (HRP-) conjugated secondary antibodies at room temperature for 1 h. The primary antibodies used in this study were listed as follows: anti-Rap1GAP (1 : 10000, Abcam, Cambridge, UK), anti-LC3B I/II (1 : 1000; Cell Signaling Technology, MA, USA), anti-P62 (1 : 1000; Cell Signaling Technology), anti-Akt and anti-p-Akt (1 : 1000; Cell Signaling Technology), anti-mTOR and anti-p-mTOR (1 : 1000; Cell Signaling Technology), anti-P70s6k and anti-p-P70s6K (1 : 1000; Cell Signaling Technology), and GAPDH (1 : 1000; Cell Signaling Technology). Protein levels were normalized to GAPDH, and the results of signals were quantified by the software of ImageJ.

Gao Y., Zhao D., Xie W.Z., Meng T., Xu C., Liu Y., Zhang P., Bi X, & Zhao Z. (2021). Rap1GAP Mediates Angiotensin II-Induced Cardiomyocyte Hypertrophy by Inhibiting Autophagy and Increasing Oxidative Stress. Oxidative Medicine and Cellular Longevity, 2021, 7848027.

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Western Blot Analysis of Cellular Stress

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Cell lysates were standardized for protein content, resolved on 4% to 12% SDS polyacrylamide gels, and blotted onto nitrocellulose membranes. Membranes were probed with rabbit anti-pPKM2, anti-PKM2, anti-CHOP, anti-GRP78/BIP, anti-pEGFR (Y1086), anti-EGFR, anti-LC3B-I/II and anti-β-actin (Cell Signaling). Antibody binding was detected by using an ECL Chemiluminescence Kit (Thermo Scientific).

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Molecular Assays for Prostate Cancer

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MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent, propidium iodide (PI) and dihydrotestosterone (DHT) were purchased from Sigma (Taufkirchen, Germany). For the protein detection the following antibodies were used: anti-AR-FL (Santa Cruz, sc-816, 1:200), anti-AR-V7 (abcam, #198394, 1:1000), anti-BAD (Cell Signaling, #9239, 1:1000), anti-Bax (Cell Signaling, #5023, 1:1000), anti-Bcl-2 (Cell Signaling, #2876, 1:1000), anti-Bcl-xL (Cell Signaling, #2764, 1:1000), anti-LC3B-I/II (Cell Signaling, #2775, 1:1000), anti-rabbit IgG-HRP (Cell Signaling, #7074, 1:5000), anti-mouse IgG-HRP (GE Healthcare, NXA931, 1:10000), anti-β-Actin-HRP (Santa Cruz, sc-1616, 1:200), anti-α-Tubulin (Sigma-Aldrich, T5168, 1:5000).

Dyshlovoy S.A., Otte K., Tabakmakher K.M., Hauschild J., Makarieva T.N., Shubina L.K., Fedorov S.N., Bokemeyer C., Stonik V.A, & von Amsberg G. (2018). Synthesis and anticancer activity of the derivatives of marine compound rhizochalin in castration resistant prostate cancer. Oncotarget, 9(24), 16962-16973.

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Antibody Panel for Prion Protein

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Mouse monoclonal anti-PrP antibody (clone 3F4) was purchased from Signet Lab, London, UK. Rabbit polyclonal anti-PrP (clone FL 253) and anti-cathepsin-D (active fragment) were purchased from SantaCruz, CA, USA. Anti-LC3BI-II and anti-P62 were purchased from Cell Signaling Technology. Anti-β-actin and anti-α-tubulin mouse monoclonal antibodies were from Sigma-Aldrich, Milano, Italy.

Thellung S., Scoti B., Corsaro A., Villa V., Nizzari M., Gagliani M.C., Porcile C., Russo C., Pagano A., Tacchetti C., Cortese K, & Florio T. (2018). Pharmacological activation of autophagy favors the clearing of intracellular aggregates of misfolded prion protein peptide to prevent neuronal death. Cell Death & Disease, 9(2), 166.

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Autophagy Regulation of L02 Cells

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L02 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and maintained in RPMI 1640 medium (Gibco, 31800022) supplemented with 10% foetal bovine serum (FBS; Gibco, 10100147), 1.5 g/L NaHCO₃ (Gibco, 25080094), and 0.11 g/L sodium pyruvate (Gibco, 11360070) in 5% CO₂ at 37°C. Emodin (E7881), 3-methyladenine (M9281) and rapamycin (V900930) were purchased from Sigma-Aldrich Chemical. The antibodies used were as follows: anti-cleaved caspase-3 (#9664), anti-LC3B-I/II (#3868), anti-phospho-AKT(Thr308) (#13038), anti-AKT (#4691), anti-phospho-mTOR(Ser2448) (#5536), anti-mTOR (#2983), and anti-actin (#8457), which were purchased from Cell Signalling Technology. Anti-phospho-PI3K(Tyr485) (sc-130211) and anti-PI3K (sc-365404) were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated species-specific secondary antibodies (4741506, 4741806) were purchased from Kirkegaard and Perry Laboratories.

Zheng X.Y., Yang S.M., Zhang R., Wang S.M., Li G.B, & Zhou S.W. (2019). Emodin-induced autophagy against cell apoptosis through the PI3K/AKT/mTOR pathway in human hepatocytes. Drug Design, Development and Therapy, 13, 3171-3180.

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Apoptosis and ER Stress Pathway Assay

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PG545 was generously provided by Zucero Therapeutics (Melbourne, Australia). Cisplatin was purchased from EMD Millipore (Calbiochem, Millipore, Billerica, MA) and paclitaxel (30 mg/5ml) from Hospira (Lake Forest, IL). All compounds were dissolved in phosphate buffer saline (PBS, Gibco, USA). Primary antibodies anti-cleaved PARP, anti-LC3BI/II, anti-phospho- and total-PERK, anti-Bip, anti-CHOP, anti-GAPDH, anti-IRE1α, anti-phospho- and total-ERK, anti-phospho- and total-AKT and secondary rabbit and mouse IgG were purchased from Cell Signaling Technology (Danvers, MA); anti-PCNA, anti-p62 and anti-Calnexin were purchased from Santa Cruz (Santa Cruz, CA).

Hoffmann R., Bhattacharya S.S., Roy D., Winterhoff B., Schmidmaier R., Dredge K., Hammond E, & Shridhar V. (2020). Sulfated glycolipid PG545 induces endoplasmic reticulum stress and augments autophagic flux by enhancing anticancer chemotherapy efficacy in endometrial cancer. Biochemical pharmacology, 178, 114003.

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Immunoblotting Analysis of Apoptosis and Autophagy

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After each experimental point, HGFs were washed with PBS, collected and lysed in RIPA buffer (Pierce, Life technologies, Milan, Italy) containing protease inhibitor cocktail. Proteins (50μg/sample) were separated by 4-12% SDS/PAGE and transferred to a nitrocellulose membrane (GE Healthcare Europe GmbH, Milan, Italy). After being blocked with 5% skim milk powder diluted in TBS containing 5% Tween-20 for 30 min, the membrane was incubated with a primary antibody, anti-caspase -3 (Cell Signaling Technology, Danvers, MA, USA), anti- beclin -1 (Cell Signaling Technology, Danvers, MA, USA), anti-LC3B I-II (Cell Signaling Technology, Danvers, MA, USA), anti-PARP (Santa Cruz Biotecnology, Inc, Santa Cruz, California), and anti β-tubulin (Sigma Aldrich, St. Louis, Missouri, USA) at 4°C overnight. Immunoreactive proteins were detected using an enhanced chemiluminescence light (ECL) detecting kit (GE Healthcare Europe GmbH, Milan, Italy). β-tubulin acted as a loading control. Images were obtained by Image Station 2000R (Kodak, New York, New York). The experiments were replicated at least three times, and representative results are shown.

Teti G., Orsini G., Salvatore V., Focaroli S., Mazzotti M.C., Ruggeri A., Mattioli-Belmonte M, & Falconi M. (2015). HEMA but not TEGDMA induces autophagy in human gingival fibroblasts. Frontiers in Physiology, 6, 275.

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Western Blot Analysis of Cellular Proteins

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The samples were homogenized in 0.1% SDS buffer containing 10 mM EDTA, 125 mM NaCl, 25 mM HEPES, 0.5% deoxycholic acid, 10 mM Na₃VO₄, 0.1% SDS, 1% Triton X-100 with Complete™ protease inhibitor cocktail (Roche, Basel, Switzerland). The cell lysate was centrifuged at 12,000 rpm for 15 min. Then the supernatant-contained protein was collected and the protein concentration was tested by protein assay kit (Bio-Rad, CA, USA). The collected protein was separated on SDS-PAGE gel, and transferred onto PVDF membrane (Millipore, MA, USA). The membrane was blocked with 5% skim milk for 1 h to reduce non-specific binding. Then, the membrane was incubated with one of the following rabbit polyclonal primary antibodies: anti-LC3B-I&II, anti-Beclin-1, anti-p62, anti-β-actin, anti-Tublin, anti-C-myc, anti-N-myc, anti-L-myc, anti-GLS and anti-ERα (Cell Signaling Technology, MA, USA) at 4 °C for 12 h. After 3 times of washes, the blot was incubated with secondary antibody HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, USA) for 1 h at room temperature. Finally, the signal was detected by the enhanced chemiluminescence kit (Biorbyt, CA, USA) and exposed to X-film

Zhou W.J., Zhang J., Yang H.L., Wu K., Xie F., Wu J.N., Wang Y., Yao L., Zhuang Y., Xiang J.D., Zhang A.J., He Y.Y, & Li M.Q. (2019). Estrogen inhibits autophagy and promotes growth of endometrial cancer by promoting glutamine metabolism. Cell Communication and Signaling : CCS, 17, 99.

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