Anti lc3b rabbit polyclonal antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Anti-LC3B rabbit polyclonal antibody is a laboratory tool used to detect the presence of the LC3B protein, which is involved in the process of autophagy. This antibody is designed to specifically recognize and bind to the LC3B protein, allowing researchers to study its expression and localization within cells.

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6 protocols using anti lc3b rabbit polyclonal antibody

Western Blotting of Autophagy Markers

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The anti-LC3B rabbit polyclonal antibody used in the western blot assay was from Cell Signaling Technology (#12741S; Beverly, MA, USA), the anti-p62/sequestosome 1 (SQSTM1) rabbit polyclonal antibody was obtained from Proteintech (#18420-1-AP; Chicago, IL, USA) and the anti-GAPDH mouse monoclonal antibody was from Beijing CoWin Biotech Co., Ltd. (cw0100A; Beijing, China). Goat anti-rabbit immunoglobulin G-horseradish peroxidase (IgG-HRP) and goat anti-mouse IgG-HRP (Pierce, Thermo Fisher Scientific, Rockford, IL, USA) were used as secondary antibodies, and enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific) was used for developing the blots. The agents epirubicin, doxycycline, 3-methyladenine (3-MA; Sigma-Aldrich, St. Louis, MO, USA) and purimycin (Invitrogen Life Technologies, Carlsbad, CA, USA) were dissolved in phosphate-buffered saline (PBS) to form stock solutions and then added directly into the media to the required concentration.

PENG W., DU T., ZHANG Z., DU F., JIN J, & GONG A. (2015). Knockdown of autophagy-related gene LC3 enhances the sensitivity of HepG2 cells to epirubicin. Experimental and Therapeutic Medicine, 9(4), 1271-1276.

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Immunofluorescence of Autophagy Marker LC3B

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1 × 106 cells were seeded on 6-well plates, each well containing a sterile glass coverslip coated with poly-L-lysine. Untreated and edelfosine-treated cells were fixed in formaldehyde (4% in PBS) for 20 min at room temperature. After fixation, cells were permeabilized in cold PBS containing 0.1% Triton X-100 for 1 min and rinsed thoroughly with PBS. Staining was performed incubating the coverslip with a specific anti-LC3B rabbit polyclonal antibody (Cell Signaling Technology) (1:100 dilution) for 1-h at room temperature, followed by 1-h at room temperature with CY3-conjugated anti-rabbit immunoglobulin (Ig) antibody (diluted 1:150 in PBS; Jackson ImmunoResearch, West Grove, PA), and DAPI staining (0.5 μg/ml; 5 min). Each incubation was followed by 2 washes in PBS. Stained coverslips were then mounted on slides using the antifading reagent SlowFade Gold (Invitrogen, Eugene, OR) to preserve fluorescence signal intensity. Samples were analyzed by microscopy using a confocal Leica SP5 microscope and LAS AF software.

Melo-Lima S., Celeste Lopes M, & Mollinedo F. (2014). Necroptosis is associated with low procaspase-8 and active RIPK1 and −3 in human glioma cells. Oncoscience, 1(10), 649-664.

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Western Blot Analysis of Autophagy Markers

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Cells were assigned to control, H₂O₂ and H₂O₂ + MP groups as described above. After lysis, protein concentrations were measured using the bicinchoninic acid method. Lysates were electrophoresed on 5% stacking gel at 90 V, and 10% separating gel at 120 V, then wet-transferred onto polyvinylidene fluoride membranes at 350 mA for 1 hour 35 minutes, after immersing the dry membranes in methanol for 30 seconds and transfer buffer for 10 minutes. Membranes were blocked with 1% bovine serum albumin for 2 hours, and incubated with rabbit anti-LC3B polyclonal antibody (1:1,000; Cell Signaling, China), mouse anti-β-actin polyclonal antibody (1:1,000; Abcam), and rabbit anti-Beclin-1 polyclonal antibody (1:1,000; Abcam) at 4°C overnight. The next day, the membranes were rinsed in Tris-buffered saline with Tween-20 (TBST), three times for 10 minutes each time, incubated with goat anti-rabbit or anti-mouse secondary antibody (1:1,000; Bioss, Beijing, China) at room temperature for 2 hours, then washed with TBST as before. Proteins were visualized using enhanced chemiluminescence. Gray values of the protein bands (target protein/β-actin) were calculated using ImageJ2x software (National Institutes of Health, Bethesda, MD, USA).

Gao W., Chen S.R., Wu M.Y., Gao K., Li Y.L., Wang H.Y., Li C.Y, & Li H. (2016). Methylprednisolone exerts neuroprotective effects by regulating autophagy and apoptosis. Neural Regeneration Research, 11(5), 823-828.

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Immunofluorescence Analysis of LAMP and LC3B

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For immunofluorescence labeling cells were cultured on 24 wells culture plates containing glass coverslips at a seeding density of 1×10⁴. Briefly, HepG2 cells were fixed for 15 minutes with 4% paraformaldehyde and washed twice with PBS 1X. Cells were blocked and permeabilized with PBS 1X + 0.2 % saponin and 1% fatty acid free BSA (FFA-BSA) for 15 minutes at room temperature. After washing twice with PBS 1X, cells were incubated with a rat anti-LAMP monoclonal antibody [GL2A7] (Abcam) and rabbit anti- LC3B polyclonal antibody (Cell Signaling) diluted both 1:300 in 1X PBS with 1 % ffa-BSA O/N at 4°C and washed twice with PBS 1X followed by incubation with a secondary anti-rat IgG antibody, conjugated to Alexa 488 (1:500) and anti-rabbit IgG antibody, conjugated to Alexa 647 (1:500) for 1 hour at 25°C. Coverslips were washed twice with PBS 1X and mounted on glass slides with fluorescent mounting medium Fluoroshield^™ with DAPI (Sigma) and visualized in a Leica SPE confocal laser-scanning microscope.

Ordoñez R., Fernández A., Prieto-Domínguez N., Martínez L., García-Ruiz C., Fernández-Checa J.C., Mauriz J.L, & González-Gallego J. (2015). Ceramide metabolism regulates autophagy and apoptotic-cell death induced by melatonin in liver cancer cells. Journal of pineal research, 59(2), 178-189.

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Immunohistochemical Analysis of Dopaminergic Neurons

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The following primary antibodies were used in this study: rabbit polyclonal anti-TH antibody (Calbiochem, #657012); chicken polyclonal anti-TH antibody (Abcam, ab76442); rabbit polyclonal anti-LC3B antibody (Cell Signaling, #2775); rabbit polyclonal anti-vesicular monoamine transporter 2 antibody (Abcam, ab70808); rat monoclonal anti-dopamine transporter antibody (Abcam, ab5990); rabbit polyclonal anti-PIN antibody targeting mouse PLA2g6 PIN domain (encoded by exon 8b, which is present in (L), but spliced out in (S) variant) were custom made by Yenzym Antibodies, LLC (San Francisco, CA), rabbit polyclonal anti-LC3B antibody (MBL, PD014); and monoclonal anti-β-actin antibody (Sigma, A1978). Secondary antibodies are the following: goat anti-rabbit Alexa488 (Invitrogen A11034), goat anti-chicken Alexa594 (Abcam ab150172) and goat anti-rat Alexa647 (Abcam ab150167) were used for imaging; HRP-conjugated anti-rabbit (Dako, K4002) was used for 3,3′-diaminobenzidine staining; HRP-conjugated anti-mouse (Cell Signaling, #7076) or anti-rabbit IgG (Cell Signaling, 7074) was used as secondary Ab for western blots.

Zhou Q., Yen A., Rymarczyk G., Asai H., Trengrove C., Aziz N., Kirber M.T., Mostoslavsky G., Ikezu T., Wolozin B, & Bolotina V.M. (2016). Impairment of PARK14-dependent Ca2+ signalling is a novel determinant of Parkinson's disease. Nature Communications, 7, 10332.

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Oxidative Stress and Mitochondrial Dynamics

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The analytical-grade reagents used in the extraction process were purchased from RCI Labscan (Bangkok, Thailand). The 2,7-dihydrofluorescein diacetate (H₂DCF-DA) was obtained from Thermo Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle medium (DMEM), sodium selenite, chloroquine, 4′,6-diamidino-2-phenylindole (DAPI) and L-glutamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Biobasic (Markham, ON, Canada). CytoTox 96 ^® Non-Radioactive Cytotoxicity assay was purchased from Promega (Madison, WI, USA). Alexa Fluor 488, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mouse monoclonal anti-β-actin antibody, mouse monoclonal anti-rabbit IgG, HRP-linked antibody, rabbit polyclonal anti-LC3B antibody, rabbit monoclonal anti-TOM20 (D8T4N) antibody and tetramethylrhodamine ethyl ester (TMRE) were purchased from Cell Signaling Technology (Denvers, MA, USA). All primers, the AccuPower^® RT Premix kit and AccuPower^® 2X GreenStar^TM qPCR Master Mix were purchased from Bioneer (Daejeon, South Korea). Mitotracker Red CMXRos was obtained from Molecular Probes (Eugene, OR, USA).

Vongthip W., Sillapachaiyaporn C., Kim K.W., Sukprasansap M, & Tencomnao T. (2021). Thunbergia laurifolia Leaf Extract Inhibits Glutamate-Induced Neurotoxicity and Cell Death through Mitophagy Signaling. Antioxidants, 10(11), 1678.

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