Lc3a b d3u4c

Manufactured by Cell Signaling Technology

Sourced in United States

LC3A/B (D3U4C) is an antibody product manufactured by Cell Signaling Technology. The antibody recognizes the LC3A and LC3B proteins, which are involved in the process of autophagy, a cellular mechanism for the degradation and recycling of damaged or unwanted components.

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6 protocols using lc3a b d3u4c

Autophagy Regulation and DNA Repair Analysis

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Olaparib (Selleckchem, AZD2281), rapamycin (LKT Labs, 53123-88-9), and bafilomycin (Sigma-Aldrich, 88899-55-2) were used. The following antibodies were used for the study: Beta-Actin (AC14) (abcam, AB6276, 1:20000 dilution); Atg16L1 (D6D5) (Cell Signaling, 8089 T, 1:1000 dilution); Atg5 (D5F5U) (Cell Signaling, 12994 S, 1:1000 dilution); Atg12 (D88H11) (Cell Signaling, 4180 S, 1:1000 dilution); Beclin-1 (D40C5) (Cell Signaling, 3495 S, 1:1000 dilution); Atg7 (D12B11) (Cell Signaling, 8558 S, 1:1000 dilution); β-Tubulin (D2N5G) (Cell Signaling, 15115 S, 1:1000 dilution); Filamin A (Cell Signaling, 4762 S, 1:1000 dilution); SP1 (Sigma, PLA0307, 1:5000 dilution); anti-phospho-Histone H2A.X (Ser139) (Sigma-Aldrich, JBW301, 1:2000 dilution); LC3 A/B (D3U4C) (Cell Signaling, 12741 S, 1:750 dilution); phospho-mTOR (Ser2448) (D9C2) (Cell Signaling, 5536 T, 1:1000 dilution); SQSTM1/p62 (Cell Signaling, 5114 T, 1:1000 dilution) and (abcam, ab56416, 1:100 dilution); Rad51 (114B4) (abcam, ab213, 1:750 dilution) and BRCA1 (Sigma Millipore, 07-434, 1:1000 dilution), PARP1 (proteintech, 66250, 1:650 dilution) and PAR/pADR (R&D systems, 4335-MC-100, 1:1000 dilution).

Cahuzac M., Langlois P., Péant B., Fleury H., Mes-Masson A.M, & Saad F. (2022). Pre-activation of autophagy impacts response to olaparib in prostate cancer cells. Communications Biology, 5, 251.

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Characterization of Neisseria gonorrhoeae antibodies

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The purified chicken IgY anti-Neisseria gonorrhoeae antibody was raised against formalin-killed FA1090 gonococci by Cocalico Biologicals and it cross-reacts with the MS-11, FA19 and F62 strains. The rabbit anti-N.g polyclonal antibody (cat# 20-NR08) was purchased from Fitzgerald Industries. Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling—Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S). Mouse antibodies against the following antigens were purchased from Santa Cruz–Actin (sc8432), FMNL1 (sc390466), FMNL2 (sc390298), FHOD1 (sc365473), DAAM1 (sc100942). Mouse anti-Cas9 (7A9-3A3) was purchased from Cell Signaling. Anti-FMNL3 (ab57963) and anti-Galectin 3 (ab2785) mouse monoclonal antibodies were purchased from Abcam. Anti-human LAMP-2 (H4B4) was purchased from BioLegend. Anti-DIAPH2 (A300-079A-T) was purchased from Bethyl Labs. Highly cross-absorbed Alexa fluorophore conjugated secondary antibodies were purchased from Life Technologies.
Inhibitors used in this study were purchased from: (1) Cayman Chemicals—cytochalasin D (5μM), CK869 (10μM), Dynasore (30μM), GSK269962 (1μM), Nocodazole (3μM), ML-141 (5μM), EHT 1864 (5μM), BMS-5 (1μM) and (-) blebbistatin (5μM); (2) Cell Signaling—LY294002 (10μM); (3) Abcam—SMIFH2 (12.5μM to 100μM).

Ivanov S.S., Castore R., Juarez Rodriguez M.D., Circu M, & Dragoi A.M. (2021). Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages. PLoS Pathogens, 17(12), e1010184.

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Immunoblotting of Cell Signaling Proteins

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The samples were analysed by SDS-PAGE electrophoresis, loading equal amounts of homogenate on 12.5% polyacrylamide Tris-glycine gels, and transferring to a nitrocellulose membrane (Amersham, GE Healthcare Europe GmbH, Milano, Italy). Specific antibodies were employed to reveal protein levels by immunoblotting: rabbit polyclonal anti-claudin2 (1:250) (#51-6100 Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA); rabbit polyclonal LC3A/B (D3U4C) (1:1000) (#12741 Cell Signaling Technology); rabbit polyclonal p62 (1:1000) (#39749 Cell Signaling Technology); and rabbit polyclonal anti-β-actin (1:1500) (A2066 Sigma-Aldrich, St. Louis, MI, USA). Immunoreactive proteins were revealed by enhanced chemiluminescence (ECL) and semi-quantitatively estimated using an LAS800 Image Station. Normalization in the same sample was carried out with respect to the β-actin homogenate samples [52 (link),53 (link)].

Lonati E., Sala G., Corbetta P., Pagliari S., Cazzaniga E., Botto L., Rovellini P., Bruni I., Palestini P, & Bulbarelli A. (2023). Digested Cinnamon (Cinnamomum verum J. Presl) Bark Extract Modulates Claudin-2 Gene Expression and Protein Levels under TNFα/IL-1β Inflammatory Stimulus. International Journal of Molecular Sciences, 24(11), 9201.

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Libertellenone C Protects Against Inflammation

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Libertellenone C (LC) was obtained as a white powder from the fermentation products of A. arundinis, which was analyzed by HR-MALDI mass spectrometry for the molecular formula C₂₀H₂₈O₅ (obsd [M + Na]⁺ at m/z 371.1829, calcd [M + Na]⁺ 371.1834). LC was dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich, MO, USA), with a stock concentration of 40 mM. H₂O₂ and sodium glutamate were purchased from RHAWN reagent (Shanghai, China). Antibodies against p-NF-κB p65 (SC-136548) and NF-κB p65 (SC-8008) were obtained from Santa Cruz Biotecnology, USA. Antibodies against NLRP3 (D2P5E, #13158), ASC (D2W8U, #67824), Caspase-1 (E2Z1C, #24232), iNOS (D6B6S, #13120), COX-2 (D5H5, #12282), HO-1 (E8B7A, #26416), and LC3A/B (D3U4C, #12741) were obtained from CST (Cell Signaling Technology, MA, USA). Antibodies against β-actin (Ab8226) were obtained from Abcam (Cambridge, UK).

Cao J., Li L., Zhang R., Shu Z., Zhang Y., Sun W., Zhang Y, & Hu Z. (2024). Libertellenone C attenuates oxidative stress and neuroinflammation with the capacity of NLRP3 inhibition. Natural Products and Bioprospecting, 14(1), 17.

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Glioblastoma and HEK293 Cell Culture

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Human glioblatoma cell lines U251 and Human Embryonic Kidney (HEK) 293 cells were maintained in DMEM medium with high glucose and sodium pyruvate, supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Human glioblatoma cell lines U87 were maintained in MEM medium supplemented with 10% fetal bovine serum. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. Cells were authenticated that it origins from ATCC by short-tandem repeat profiling. Antibodies against HIF1α(20960-1-AP), GST(66001-1-Ig), and HA(51064-2-AP) were purchased from ProteinTech. Antibodies against FIH1(sc-26219) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FLAG (F1804) was purchased from Sigma-Aldrich. Antibodies against CA9 (No. D120346), GLUT1 (No. D160433), LDH-A (No. D199841), and PHD2 (No. D122872) were purchased from Sangon Biotech. Antibodies against ATG5 (D5F5U), ATG7 (D12B11), ATG12 (D88H11), and LC3A/B (D3U4C) were purchased from Cell Signaling Technology.

Feng J., Zhang Y., She X., Sun Y., Fan L., Ren X., Fu H., Liu C., Li P., Zhao C., Liu Q., Liu Q., Li G, & Wu M. (2018). Hypermethylated gene ANKDD1A is a candidate tumor suppressor that interacts with FIH1 and decreases HIF1α stability to inhibit cell autophagy in the glioblastoma multiforme hypoxia microenvironment. Oncogene, 38(1), 103-119.

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Antibody Panel for Autophagy Signaling

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The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): LC3A/B (D3U4C) (#12741), total 4E-BP1 (53H11) (#9644), phospho-4E-BP1 (Thr37/46) (#2855), total p70-S6K (49D7) (#2708), phospho-p70-S6K (Thr389) (#9205), total mTOR (#2983), phospho-mTOR (Ser2448) (#1356), Alix (3A9) (#2171), Flotillin-1 (D2V7J) (#18,634), β-actin (13E5) (#4970) for immunofluorescence, α-tubulin (DM1A) (#3873) for immunofluorescence. Other antibodies used: LAMP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-19992), M6PR (Abcam, Cambridge, UK, ab32815) for immunofluorescence, M6PR (BBI Life Sciences, Shanghai, China, D154247) for western blotting, CD63 (BioLegend, San Diego, CA, USA, 143,902), Cathepsin D (Abcam, ab75852), Vinculin (PTM Biolabs, Hangzhou, China, PTM-5168), β-Actin (Ruiying Biological, Suzhou, China, RLT0099) for immunoblotting.

Sun S., Li Q., Liu G., Huang X., Li A., Guo H., Qi L., Zhang J., Song J., Su X, & Zhang Y. (2024). Endosomal protein DENND10/FAM45A integrates extracellular vesicle release with cancer cell migration. BMC biology, 22(1).

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