Total protein was extracted from cultured cochlear tissue (n = 10 per group). Protein concentrations were determined using a bicinchoninic acid (BCA) protein kit (Beyotime, P0010S). Protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then blotted onto polyvinylidene fluoride (PVDF) membranes (Immobilon-140 P; Millipore, Bedford, MA). The membranes were blocked (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; and 0.1% Tween-20) for 1 h at room temperature and incubated overnight at 4°C with either anti-LC3B (1:1000 dilution; Sigma-Aldrich, L7543) or anti-β-actin (1:10,000 dilution; Cell Signaling Technology, abs119600) primary antibodies. The membranes were washed three times in Tris-buffered saline with Tween-20 (TBST) for 10 min each and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilution; Cell Signaling Technology, A0208) for 1 h at room temperature. Blots were developed using an enhanced chemiluminescence (ECL)-Kit (Beyotime, Jiangsu, China). Each experiment was repeated at least three times.
Anti lc3b
Manufactured by Merck Group
Sourced in United States, United Kingdom, France
Anti-LC3B is a laboratory reagent that can be used for the detection and quantification of the LC3B protein, which is a commonly used marker for autophagy. It is a highly specific antibody that binds to the LC3B protein and can be used in various immunoassay techniques, such as Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (10 mentions)
Anti lamp2, by Santa Cruz Biotechnology (5 mentions)
Image lab software, by Bio-Rad (5 mentions)
Anti actin, by Merck Group (5 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Anti phospho akt, by Cell Signaling Technology (4 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Anti p62, by Abcam (4 mentions)
Anti actb, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti ulk1, by Cell Signaling Technology (3 mentions)
Anti bax, by Cell Signaling Technology (3 mentions)
Anti beclin 1, by Cell Signaling Technology (3 mentions)
Anti atg5, by Cell Signaling Technology (3 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (3 mentions)
74 protocols using anti lc3b
1
Western Blot Analysis of Cochlear Tissue
2
Autophagy and mTOR Pathway Regulation
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The following antibodies were used: rabbit anti-mouse p62/SQSTM1 antibody (MBL International, Japan; PM045), anti-LC3B (Sigma, St Louis, MO, USA; L7543), anti-mTOR (CST, Boston, MA, USA; 7C10, anti-phospho-mTOR (Ser2448; CST; 2971S), anti-RPS6KB1 (CST; 4907), anti-Phospho-RPS6KB1 (Thr389) (CST; 9234s), anti-PIK3CAp85 antibody (CST; 4292), anti Phospho-PIK3CAp85 (Tyr458) (CST; 4228), anti-AKT antibody (CST; 9272s), anti phospho-AKT (Ser473) (CST; 4060s), anti-phospho-EIF4EBP1 (Thr37/46) (CST; 236B4) and anti-EIF4EBP1 (Abcam, Cambridge, UK, ab2606). Secondary antibodies included Alexa Fluor 488-labeled goat anti-mouse and rabbit IgG(H+L) (ZSGB-BIO; ZF-0512 and ZF-0511), Alexa Fluor 594-labeled goat anti-mouse and rabbit IgG(H+L) (ZSGB-BIO; ZF-0513 and ZF-0516), DyLight 680-conjugated anti-mouse and rabbit IgG (H&L) (Rockland, Limerick, PA, USA; 610-144-002 and 611-144-002), DyLight 800-conjugated anti-mouse and Rabbit IgG (H&L) (Rockland; 610-145-002 and 611-145-002). Other reagents used in this study were: Dual-Luciferase Reporter (DLR) Assay System (Promega, Madison, WI, USA; E1910), Hoechst 33342 (Life, Carlsbad, CA, USA; H1399), CQ (Sigma-Aldrich, St Louis, MO, USA; c6628), 3-MA (NSC 66389) and Perifosine (Selleck, Houston, TX, USA, S1037).
3
Immunoblotting Analysis of Cellular Signaling Pathways
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Cellular lysates were prepared using lysis buffer (20 mM MOPS pH7.4, 100 mM KCl, 1 mM DTT, 1 mM EDTA, 2 mM benzamidine, 25 mM NaF, 5 μg/mL leupeptin, 10 mM chymostatin, 1 μM microcystin LR, 1 X EDTA-free protease inhibitor cocktail (Roche)). The concentration of lysate protein was determined by Bradford assay (Bio-Rad). Immunoblotting was performed using the Mini-PROTEAN Tetra Cell System (Bio-Rad) with 20 μg of lysate protein. The primary antibodies used were β-actin (Abcam), cleaved PARP (Cell Signalling Technologies), AMPK(phospho-T172; Cell Signalling Technologies), eIF4E-(phospho-S209; Abcam), S6K(phospho-T389; Cell Signalling Technologies), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204, Cell Signalling Technologies), 4E-BP1 (Cell Signalling Technologies), p38-MAPK (Thr180/Tyr182, Cell Signalling Technologies), Phospho-Mnk1 (T197/202; Cell Signalling Technologies), Phospho-S6 Ribosomal Protein (S240/244, Cell Signalling Technologies), eIF4E (Cell Signalling Technologies), Phospho-Akt (T308, Abcam), Phospho-ULK1 (S555, Cell Signalling Technologies) and Anti-LC3B (Sigma).
4
Western Blot Analysis of Viral and Cellular Proteins
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Precipitated or cell lysate proteins were separated by 8 to 12% SDS-PAGE, and samples were then transferred to polyvinylidene difluoride membranes (GE Health). Subsequently, these proteins were detected with the following antibodies: anti-FLAG-M2 (Sigma; 0.5 mg/ml; 1:1,000), anti-PB2 (Santa Cruz; 1:500), anti-TUFM (Santa Cruz; 1:800), anti-actin (Millipore; 1:4,000), anti-COX4 (GeneTex; 1:3,000), anti-calreticulin (GeneTex; 1:1,500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abnova; 1:5,000), anti-lamin B1 (Abcam, Inc.; 1:4,000), anti-LC3B (Sigma; 1:2,500), anti-α-tubulin (Abcam, Inc.; 1:6,000), anti-Myc (Sigma; 1:1,500), anti-PB1 (GeneTex; 1:2,500), anti-PA (GeneTex; 1:2,000), anti-NP (generated by our colleague Cheng-Kai Chang; 1:10,000), anti-NLRX1 (Millipore; 1:100), anti-Atg12 (GeneTex; 1:1,500), and anti-Atg16L1 (GeneTex; 1:1,500).
5
Western Blot Analysis of Autophagy and Inflammation
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Protein samples were boiled for 5 min in 5× SDS-PAGE loading buffer and loaded onto 10–15% SDS polyacrylamide gels. Proteins were transferred to PVDF membranes, and membranes were blocked for 2 h in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 with 5% non-fat dry milk and then incubated at 4° C overnight with the following primary antibodies: anti-LC3B (1:2,000; Sigma-Aldrich), anti-P62 (1:5,000; Proteintech), anti-P65 (1:1,000; Cell Signaling Technology), anti-phospho-P65 (1:1,000; Cell Signaling Technology), anti-ICAM-1 (1:200; Santa Cruz Biotechnology), anti-PI3K (1:1,000; Cell Signaling Technology), anti-phospho-PI3K (1:1,000; Cell Signaling Technology), anti-Akt (1:1,000; Cell Signaling Technology), anti-phospho-Akt (1:1,000; Cell Signaling Technology), anti-phospho-p70S6K (1:1,000; Cell Signaling Technology), or anti-GAPDH(1:50,000; Proteintech). Blots were washed and then incubated with the HRP-conjugated secondary antibody (1:10,000; Proteintech), and blots were revealed using ECL. ImageJ software was used for the semi-quantification of protein expression.
6
Protein Localization and Autophagy Markers
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The following primary antibodies were used: anti-GAPDH (Sigma-Aldrich, G9545; 1:5000) anti-TOMM20 (Santa Cruz Biotechnology, SC-11415; 1:500), anti-FIS1 (Proteintech, 10956–1-1ap; 1:100), anti-ATP5F1A/ATP5A (Abcam, Ab14748; 1:200), anti-PDHA1 (Abcam, ab110330; 1:200), anti-ATG7 (Cell Signaling Technology, 8558; 1:1000), anti-RAB9A (Cell Signaling Technology, 5118; 1:1000), anti-ULK1 (Cell Signaling Technology, 8054; 1:1000), anti-RAB5A (Cell Signaling Technology, C8B1; 1:1000), anti-RAB7 (ERP7589; Abcam, ab137029; 1:1000), anti-LC3B (Sigma-Aldrich, L7543;1:200). Alexa Fluor 647‐conjugated goat anti-rabbit and anti-mouse IgG (Invitrogen, A21244 and A32728; 1:500) were used as secondary antibodies.
7
Antibody Usage for Autophagy Analysis
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The following commercially available antibodies were used: anti-GABARAP for Drosophila Atg8a (PM037, MBL), anti-Ref(2)P (ab178440, Abcam), anti-Tau 5A6 (5A6, DSHB), anti-Phospho-Tau AT8 (MN1020, Thermo), anti-cleaved caspase 3 (9661, Cell Signaling), anti-LC3B (L7543, Sigma), anti-p62 (NBP1-48320, Novus Biologicals), anti-GFP (G5144, Invitrogen), anti-Actin (A1978, Sigma), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (Thermo Fisher Scientific), or near infrared (IR) dye 700/800 (Rockland).
8
Cardiac Injury Biomarker and Apoptosis Assays
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Met, CQ, TTC, Evans Blue, dimethyl sulfoxide (DMSO), TMRE, MDC, PI, Hoechst was obtained from Sigma-Aldrich (St. Louis, MO, USA). Miransertib was purchased from Selleck Chemicals (Houston, Texas, USA).DMEM and FBS were purchased from Gibco Laboratories (Life Technologies, Inc., Burlington, ON, Canada).CCK-8 was purchased from Dojindo Laboratories (Tokyo, Japan).The commercial assay kits for the CK-MB were from Jiancheng Bioengineering (Nanjing, Jiangsu, China). The primary antibodies used were anti-IL-1β, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Bioworld (MN, USA), anti-IL-6 (Abcam, Cambridge, MA, USA), anti-Bax, anti-phospho-Akt, anti-Akt, anti-phospho-mTOR and mTOR, anti-phospho-AMPK and AMPK, anti-Beclin-1, anti-P62, anti-Atg5 were purchased from Cell Signaling Technology (Beverly, MA, USA). In addition, anti-LC3B from Sigma and anti-Bcl-2 from Absin were used. All the antibodies were from rabbits and dilutions of antibodies were 1:1000, except anti-GAPDH (1:5000).
9
Western Blot Analysis of Cellular Proteins
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Cell pellets were resuspended in Laemmli sample buffer (BioRad), and Western blots were performed as described previously [45 (link)]. The membranes were incubated with the following primary antibodies: anti-lamin A/C (Thermo Fisher, 1/5000), anti-prelamin A (Santa Cruz, 1/3000), anti-53BP1 (Bethyl, 1/3000), anti-Rad51 (NBP2-32622, Novus Biological, 1/1000), anti-Lamp2 (Santa Cruz, 1/1000), anti-p62 (NovusBio, 1/3000), anti-LC3B (Sigma-Aldrich, 1/10000), and anti-β-actin (Sigma-Aldrich, 1/10000). Afterwards, the membranes were incubated with a corresponding secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories). Proteins were visualized with a chemiluminescence detection system (ECL substrate; BioRad) and signals were analyzed using the IMAGE LAB software (BioRad). Protein signals were quantified by normalizing to β-actin, as indicated.
10
Visualizing Autophagic Vesicles in Cells
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Epithelial cells were grown on glass cover slips prior to transfection. After knockdown of indicated genes, cells were starved for 2 hours in Hank's Balanced Salt Solution (HBSS; Sigma-Aldrich). Cells were fixed with ice-cold 100% methanol, and stained for endogenous LC3 using anti-LC3B (Sigma-Aldrich). Secondary antibodies used were Alexafluor 488 (Molecular Probes). Nuclei were stained with 1× PBS containing 0.5 µg/ml 4′,6 diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and mounted on slides with Fluoromount (Southern Biotech). Staining was visualised by an Olympus BX61 fluorescence microscope.
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