Immunoblotting was performed using anti-phospho-RB (Cell Signaling, Danvers, MA, USA), anti-p62 (GeneTex, Irvine, CA, USA), anti-LC3B (Novus biologicals, Littleton, CO, USA), anti-caspase3 (Novus biologicals), anti-γH2AX (Cell Signaling), anti-PIK3C3 (GeneTex) and anti-GAPDH (GeneTex) antibodies, followed by visualization using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence detection system.
Anti lc3b
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
Anti-LC3B is a primary antibody used for the detection of the LC3B protein. LC3B is a marker for autophagy, a cellular process involved in the degradation and recycling of damaged organelles and proteins. The Anti-LC3B antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the dynamics and regulation of autophagy in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti beclin 1, by Novus Biologicals (5 mentions)
Anti atg5, by Novus Biologicals (4 mentions)
Anti flag, by Merck Group (3 mentions)
Anti p62, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Thermo Fisher Scientific (3 mentions)
Anti p62, by Abcam (3 mentions)
Anti gapdh, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti sqstm1 p62, by Abcam (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Anti p mtor, by Cell Signaling Technology (2 mentions)
Anti atg7, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti p62 sqstm1, by Novus Biologicals (2 mentions)
Anti lamp1, by Novus Biologicals (2 mentions)
Anti apg7 atg7, by Santa Cruz Biotechnology (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
41 protocols using anti lc3b
1
Immunoblotting of Autophagy and Apoptosis Markers
2
Baicalin and 3-MA Modulate Autophagy and Apoptosis
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Baicalin and 3-MA were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). BAPTA-AM was purchased from Selleck Chemicals (Shanghai, China). Anti-Bax, anti-Bcl-xl, anti-cleaved caspase 3, anti-PARP, anti-mTOR, anti-p-mTOR, anti-AKT, anti-p-AKT, anti-cyclin D1, anti-cyclin B1, anti-cyclin A, anti-ZO-1, anti-Catenin, anti-Vimentin, and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-LC3B, anti-MMP-2, and anti-beclin 1 antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Anti-HMGB1 and anti-p62/SQSTM1 antibodies were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated and FITC-conjugated antirabbit or antimouse IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
3
Quantifying Autophagy Proteins in Cells
4
Antibody-mediated Autophagy Regulation
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Antibodies used were as follows: anti-118Y-p-paxillin, anti-31Y-p-paxillin and anti-397Y-p-FAK (Invitrogen); anti-GFP, anti-β-actin, anti-Rab7, anti-p62 and anti-LC3B (Santa Cruz); anti-416Y-p-Src and anti-NBR1 (Cell signaling), anti-GFP (Roche); anti-LAMP-1 and anti-HA (Abcam); anti-LC3B, anti-Atg12 and anti-c-Cbl (Novus); anti-Rab5a (BD transduction laboratory); anti-paxillin, anti-Src and anti-FAK (Millipore); anti-LAMP1 (R&D systems); anti-FLAG (Sigma). Anti-mouse and anti-rabbit IgG-peroxidase-conjugated secondary antibodies for Western blot assays were from Bio-Rad. Alexa Fluor594 and 488 conjugated secondary antibodies were from Life Technology. Alexa Fluor 647 conjugated secondary antibodies were from Millipore. Epidermal growth factor was from Gibco; Histodenz (Nycodenz), nocodazole and chloroquine (CQ) were from Sigma-Aldrich. When indicated CQ was used at a concentration of 20 μM, a non-toxic concentration found to induce optimal autophagy inhibition (based on changes in LC3I/II ratio), which is in accordance with previous studies (Sandilands et. al.).
5
Protein Expression Analysis in Tissues
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Snap-frozen tissues were homogenized in UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific, Waltham, MA), using FastPrep lysis tubes (2 mL, 116910500, MP Biomedicals, Irvine, CA) as described above. Protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA). SDS-page electrophoresis was performed in a 4-15% gradient polyacrylamide gel. After transfer, the membrane was blocked with Odyssey buffer (Li-Cor Biosciences, Lincoln, NE) and incubated with an anti-human GAA antibody (Cat# ab137068, RRID:AB_2687584, rabbit monoclonal, Abcam, Cambridge, MA), anti-p62 (Cat# ab56416, RRID:AB_945626 mouse monoclonal, Abcam, Cambridge, MA), anti-Vinculin (Cat# V9131, RRID:AB_477629, mouse monoclonal, Sigma Aldrich, Saint Louis, MO), anti-Gapdh (Cat# PA1-988, RRID:AB_2107310, rabbit polyclonal, Thermo Fisher, Waltham, MA), anti-Parkin (ab77924, mouse monoclonal, Abcam, Cambridge, MA), anti LC3b (Cat# sNB-100-2220, RRID:AB_10003146, rabbit polyclonal, Novus Biologicals) . The membrane was washed and incubated with the appropriate secondary antibody (Li-Cor Biosciences), and visualized by Odyssey imaging system (Li-Cor Biosciences). Band intensity was quantified by ImageJ software.
6
Cardiac Tissue Immunofluorescence for LC3B
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For immunofluorescence, frozen tissue sections from the left ventricle were stained with a rabbit polyclonal anti LC3B (Novus Biologicals, Littleton, CO). Nuclei were identified using SYTO 82 orange fluorescent nucleic acid staining (Molecular Probes, USA). The cover-glasses were mounted and sections were analyzed with a LSM 510 confocal microscopy (Zeiss).
7
Immunoprecipitation and Immunoblot Analysis
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The primary antibodies used include anti-LC3B, anti-beclin1 (Novus Biologicals, Littleton, CO, USA), anti-caveolin1 (Abcam, Cambridge, UK), anti-TIMM23, anti-phospho-caveolin1 (BD Bioscience, San Jose, CA, USA), anti-HA, anti-Vps34 (Cell Signaling Technology, Danvers, MA, USA), anti-tubulin, anti-Flag (Sigma-Aldrich), anti-p62 (Abnova, Taiwan) and anti-TOMM20 (Santa Cruz Biotechnology, Dallas, TX, USA). Immunoprecipitation assays were performed as previously described;6 (link) briefly, cells were lyzed with modified RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1% CHAPS). After pull-down with the appropriate antibodies, same amounts of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Immunoblot analysis was then performed and visualized by the enhanced chemiluminescence method.
8
Reagents and Antibodies for Cell Assays
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TSA was purchased from Enzo Life Sciences. Carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC) and 7-amino-4-trifluoromethyl coumarin (AFC) were from Enzyme Systems Products (Livermore, CA). Unless indicated, all other reagents including cisplatin and chloroquine were purchased from Sigma (St. Louis, MO). The following primary antibodies were used: anti-LC3B from Novus Biologicals (Littleton, CO); anti-ATG7, anti-β-actin and anti-cyclophilin B from Abcam; and anti-cleaved caspase3, anti-AMPK, anti-phospho-AMPK (Thr172), anti-P70S6K, anti-phospho-P70S6K (T389) from Cell Signaling Technology (Danvers, MA). All secondary antibodies for immunoblot analysis were from Thermo Scientific (Rockford, IL).
9
Western Blot Analysis of Cellular Proteins
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Cellular proteins were extracted, separated by electrophoresis, transferred to nitrocellulose membranes, and probed, as described previously [16 (link)]. The antibodies used for western blotting were anti-LC3B (Novus, NB600-1384, Saint Charles, MI, USA; Cell Signaling Technology, 12741S, Danvers, MA, USA), anti-human SQSTM1 (Abcam, ab56416; Cambridge, MA, USA), and anti-AKTser473 (Cell Signaling Technology, 9271; Danvers, MA, USA). Membranes were stained with Ponceau S Red (Sigma, P-3504; ST-Louis, MO, USA) for protein visualization. After initial probing, membranes were stripped with Restore Plus western blot stripping buffer (Thermo Fisher Scientific, 46430; Waltham, MA, USA) and then re-probed with anti-alpha-tubulin as a loading control (Cell Signaling Technology, 3873; Danvers, MA, USA). Alternatively, cellular proteins were separated by electrophoresis in stain-free gels (Bio-Rad, 456-8124; Hercules, CA, USA) for direct visualization and quantification of the proteins. Densitometric analyses were conducted with the ImageLab software from BioRad (version 1.0; Hercules, CA, USA). Data are expressed in arbitrary units.
10
Mechanistic Insights into PIWIL2-p65 Pathway Regulation
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CDS encoding PIWIL2 and p65 was synthesized and inserted into pcDNA3.1 and pEGFP, respectively. shRNA against PIWIL2 and p65 were synthesized by TSINGKE Biological Technology company (Beijing, China) and the target sequences of these shRNA were as follows:
Human PIWIL2 shRNA (shPIWIL2-1): 5′-CGG ATT GAG GAG AAA CGT AAA CTC-3′
Human PIWIL2 shRNA (shPIWIL2-2): 5′-CTA TGA GAT TCC TCA ACT ACA GAA G-3′
Human p65 shRNA (shp65): 5′ -CGG ATT GAG GAG AAA CGT AAA CTC-3′
Anti-PIWIL2, anti-p62, anti-Beclin-1, and anti-LaminB1 were purchased from Santa Cruz (USA). Anti-IKKα, anti-IKKβ, anti-p-IKKα/β(Ser176/180), anti-IκBα, anti-p-IκBα(Ser32/36), anti-p65, anti-p-mTOR(Ser2448), anti-p-ULK1(Ser555), anti-p-4E-BP1(Thr37/46), anti-p-P70S6(Thr389), and anti-TSC1 were purchased from Cell signaling technology (USA). Anti-LC3B was purchased from Novus Biologicals (USA). Anti-mTOR and anti-Bcl-2 were purchased from Proteintech Group (USA). Anti-GAPDH was purchased from Epitomics (USA).
Human PIWIL2 shRNA (shPIWIL2-1): 5′-CGG ATT GAG GAG AAA CGT AAA CTC-3′
Human PIWIL2 shRNA (shPIWIL2-2): 5′-CTA TGA GAT TCC TCA ACT ACA GAA G-3′
Human p65 shRNA (shp65): 5′ -CGG ATT GAG GAG AAA CGT AAA CTC-3′
Anti-PIWIL2, anti-p62, anti-Beclin-1, and anti-LaminB1 were purchased from Santa Cruz (USA). Anti-IKKα, anti-IKKβ, anti-p-IKKα/β(Ser176/180), anti-IκBα, anti-p-IκBα(Ser32/36), anti-p65, anti-p-mTOR(Ser2448), anti-p-ULK1(Ser555), anti-p-4E-BP1(Thr37/46), anti-p-P70S6(Thr389), and anti-TSC1 were purchased from Cell signaling technology (USA). Anti-LC3B was purchased from Novus Biologicals (USA). Anti-mTOR and anti-Bcl-2 were purchased from Proteintech Group (USA). Anti-GAPDH was purchased from Epitomics (USA).
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