The radioimmunoprecipitation (RIPA) (Beyotime Biotechnology, China) lysate was prepared by lysing tissue/cells on ice for 30 min, followed by sonication to extract the total proteins. Equivalent amounts of proteins were isolated by SDS-PAGE, followed by transfer onto nitrocellulose membranes and examination using primary antibodies. The membranes were treated with antibodies against Beclin-1, LC3II/I, BAX, BCL-2, and GAPDH (Cell Signal Technology, United States of America) and detected using an enhanced chemiluminescence reagent.
Lc3 1 2
Manufactured by Cell Signaling Technology
178 citations
Sourced in United States, China, Germany, United Kingdom
About the product
LC3-I/II is a protein marker used to detect autophagy in cells. It is a key component of the autophagosome formation process. LC3-I is a cytosolic form, and LC3-II is a lipidated form that associates with the autophagosomal membrane.
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Check compatibility
178 protocols using «lc3 1 2»
1
Western Blot Analysis of Autophagy Markers
2
Chondrocyte Inflammation and Autophagy
Dulbeccco’s Modified Eagle Medium Nutrient Mixture F-12 (C11330500BT), Penicillin Streptomycin (15140-122), and Insulin-Transferrin-selenium (ITS, 41400-045) were purchased from Gibco (Rockville, MD). The FHL2 antibody (21619-1-AP, 1:1,000 for WB, 1:100 for immunofluorescence) was purchased from Proteintech (Wuhan, Hubei, China). Antibodies against COX-2 (ab179800, 1:1,000 for WB), BAX (ab32503, 1:1,500 for WB), MMP-3 (ab52915, 1:1,000 for WB), MMP-13 (ab39012, 1:1,000 for WB), type II collagen (COL II) (ab34712, 1:1,000 for WB) were purchased from Abcam (Cambridge, UK). Antibodies against JNK (9252, 1:1,000 for WB), p-JNK (4668, 1:1,000 for WB), NF-ĸB p65 (8242, 1:1,000 for WB), p-NF-ĸB p65 (3033, 1:1,000 for WB), p38 (8690, 1:1,000 for WB), p-p38 (4511, 1:1,000 for WB), ERK1/2 (9102, 1:1,000 for WB), p- ERK1/2 (9101, 1:1,000 for WB), GAPDH (5174, 1:2,000 for WB), β-actin (4970, 1:2,000 for WB), Beclin-1 (3495, 1:1,000 for WB), LC3I/II (12741, 1:1,000 for WB) were purchased from Cell Signaling Technology (Danvers, MA). The recombinant murine IL-1β (211-11B) was purchased from Peprotech (Cranbury, NJ). The protease inhibitor cocktail (B14001) and phosphatase inhibitor cocktail (B15001) were purchased from Bimake (Shanghai, China). The RIPA buffer, WB transfer buffer, tris-glycine running buffer, and TBST buffer were purchased from Solarbio Life Sciences (Beijing, China). The electrochemiluminescence (ECL) reagent was purchased from Beijing Labgic Technology Co., Ltd. (Beijing, China).
3
Western Blot Analysis of PI3K/AKT Pathway
The proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked using 5% blocking milk, followed by incubation with primary antibodies against Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA, 1:1000), Phosphoinositide-3-Kinase Regulatory Subunit 1 (PIK3R1, 1:1000), p-AKT Serine/Threonine Kinase 1 (p-AKT1, 1:1000), BCL2 Apoptosis Regulator (BCL2, 1:1000), Autophagy-Related 5 (ATG5, 1:1000), Microtubule Associated Protein 1 Light Chain 3 Alpha/Beta (LC3I/II, 1:1000), Autophagy-Related 3 (ATG3, 1:1000), Autophagy-Related 16 Like 1 (ATG16L1, 1:1000), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, 1:40,000) for specific protein detection. The primary antibodies against PIK3CA, PIK3R1, p-AKT1, BCL2, ATG5, LC3I/II, ATG3, and ATG16L1 were purchased from Cell Signaling Technology, Danvers, MA, USA, while the primary antibody against GAPDH was purchased from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA. For Western blot quantification, the protein expression level at each concentration was first normalized to the GAPDH level of the corresponding well. The normalized value was then divided by the normalized value of the control group to obtain the final ratio.
4
Western Blot Analysis of Autophagy Markers
Western blotting analysis was conducted as previously described.
4 (link) Briefly, the total protein of samples was quantified by a BCA protein kit (Beyotime), separated on a 12% sodium dodecyl sulphate‐polyacrylamide gel (SDS‐PAGE), and transferred onto 0.45 μm polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk, the membranes were incubated with p62, Beclin‐1, LC3I/II, and GAPDH (Cell Signalling Technology, Danvers, MA) primary antibodies overnight at 4°C, and then HRP‐secondary antibodies (Beyotime). Finally, the immunoblots were analysed using enhanced chemiluminescence reagents (Bio‐Rad, Hercules, USA).
4 (link) Briefly, the total protein of samples was quantified by a BCA protein kit (Beyotime), separated on a 12% sodium dodecyl sulphate‐polyacrylamide gel (SDS‐PAGE), and transferred onto 0.45 μm polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk, the membranes were incubated with p62, Beclin‐1, LC3I/II, and GAPDH (Cell Signalling Technology, Danvers, MA) primary antibodies overnight at 4°C, and then HRP‐secondary antibodies (Beyotime). Finally, the immunoblots were analysed using enhanced chemiluminescence reagents (Bio‐Rad, Hercules, USA).
5
Quantification of Autophagy Signaling Proteins
Following the required manipulation of each experiment, tumor cells at a confluency of 70–80% were lysed using RIPA buffer (50 mM Tris-Cl pH 7.4, 1% NP-40, 150 mM NaCl, and 1 mM EDTA (purchased from Sigma-Aldrich, St. Louis, MO, USA)) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 1 mM EGTA, 10 mM NaF, and 1 mM Na3VO4 (purchased from Sigma-Aldrich, St. Louis, MO, USA)), homogenized, and incubated for 15 min on ice. Protein extracts were obtained by centrifuging at 13,000 rpm, 4 °C, for 15 min. The concentration of protein samples was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Protein samples (30 μg) were separated via SDS/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking non-specific antibody binding sites with 5% dry fat-free milk, the membrane was incubated overnight at 4 °C with primary antibodies against Hsp70 (clone 2B11), ATG5 (Cell signaling, Danvers, MA, USA #2630), LC3 I/II (Cell signaling #4108), p62 (Abcam, Cambridge, UK, #ab155686 or Servicebio, Wuhan, China, #GB11239-1-100), ULK1 total (Cell signaling #8054), p-ULK1 Ser555 (Cell signaling #5869), p-ULK1 Ser757 (Cell signaling #6888), p-AMPK (Abcam # ab133448), p-mTOR (Cell signaling #2971), β-Actin (Invitrogen, Waltham, MA, USA, #MA1-744), and β-tubulin (Invitrogen, #32-2600). After that, membranes were washed three times with PBS-T and incubated with peroxidase-conjugated secondary antibodies for 1 h. They were then washed four times with PBS-T, and then the chemiluminescence of protein bands was detected using the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).
Top 5 most cited protocols using «lc3 1 2»
1
In vivo Protein Analysis after SCI
For in vivo protein analysis, a spinal cord segment at the lesion epicentre was dissected at 7 and 14 days and immediately stored at −80°C. Proteins from animal tissue or PC12 cells were first quantified by the BCA reagent method. We separated the proteins on 8% (w/v) or 12% (w/v) gels and transferred the proteins onto polyvinylidene fluoride membrane (Bio‐Rad, Hercules, CA, USA). Membranes were blocked with 5% (w/v) milk (Bio‐Rad) in TBS with 0.05% Tween 20 (TBST) for 2 hours at room temperature and then were incubated overnight at 4°C with primary antibodies of FGF1 (1:1000; Abcam), ace‐tubulin (1:2000; Cell Signaling Technology), Tau (1:1000; Abcam), GAP43 (1:1000; Abcam), MBP (1:1000; Abcam), p‐mTOR (1:1000; Cell Signaling Technology), mTOR (1:1000; Cell Signaling Technology), ATG7 (1:1000; Bio‐world), Beclin1 (1:1000; Abcam), LC‐3 I/II (1:1000; Cell Signaling Technology), PRDX1 (1:1000; Cell Signaling Technology) and GAPDH (1:10000; Bio‐world). Next, the membranes were washed with TBST 3 times and treated with horseradish peroxidase‐conjugated secondary antibodies for 60 minutes. All signals were detected by ChemiDocXRS + Imaging System (Bio‐Rad). All experiments were repeated 3 times for accuracy.
Corresponding organizations : Second Affiliated Hospital & Yuying Children's Hospital of Wenzhou Medical University, Wenzhou Medical University, Wenzhou University
2
Protein Interactions and Signaling Pathways
Total cell lysates and cytoplasmic and nuclear extracts were isolated from the cells for Western blot assays, as described previously. [3] (link) Proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes, which were probed with primary antibodies against Drp1, Mfn2, Pink1, Nrf2, Parkin, TOM20 and VDAC (Santa Cruz Biotechnology, USA), LC3I/II, cleaved caspase-3 (Cell Signaling Technology, USA), p62 (Abcam, USA) and Keap1 (Proteintech, USA) and developed using an ECL system (Amersham Biosciences, USA). β-Actin (Santa Cruz) and histone 3 (Novus Biologicals, USA) were used as internal controls.
Each sample was adjusted to 1 mg/ml and divided into two equal aliquots containing 100 μg of protein to observe the interaction between Keap1 and Nrf2 via IP, as described previously. [7] (link) The samples were incubated with an anti-Nrf2 or anti-Keap1 antibody in an IP buffer for 12 h at 4 °C. Protein A-sepharose beads (50 μl) were added to the samples, which were incubated for another 12 h. The samples were then washed and resuspended, and Western blotting was performed as described previously. [7] (link).
Each sample was adjusted to 1 mg/ml and divided into two equal aliquots containing 100 μg of protein to observe the interaction between Keap1 and Nrf2 via IP, as described previously. [7] (link) The samples were incubated with an anti-Nrf2 or anti-Keap1 antibody in an IP buffer for 12 h at 4 °C. Protein A-sepharose beads (50 μl) were added to the samples, which were incubated for another 12 h. The samples were then washed and resuspended, and Western blotting was performed as described previously. [7] (link).
Corresponding organizations : Xiangya Hospital Central South University, Central South University, Brigham and Women's Hospital, Harvard University, Augusta University
3
Quantitative Analysis of Kidney Fibrosis
Kidney tissues from the rats were frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and sectioned at a thickness of 4 μm. The cryostat sections were fixed in acetone for 20 minutes at room temperature. Before and after incubating the tissue sections with 0.3% Triton X-100 for 5 minutes, they were washed with phosphate buffered saline (PBS) 3 times. Thereafter, tissue sections were blocked with a blocking solution containing 1% bovine serum albumin. Slides were incubated overnight at 4 °C with primary antibodies against collagen I, fibronectin (Abcam, New Territories, HK) and LC3 I/II (Cell Signaling, Beverly, MA). After washing with PBS 3 times, secondary horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulins (Abcam, New Territories, HK) were applied to the slides for 1 hour in the dark at room temperature. After washing with PBS 3 times, the slides were incubated with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) for 5–10 minutes. Using light microscopy, changes in kidneys and the positively stained areas were observed. These positive areas were visualized at a magnification of 200× and the percentages of the positive areas in whole renal areas were calculated in randomly selected 5 nonoverlapping fields with Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD).
Corresponding organizations : Nanjing University of Chinese Medicine, Nanjing Drum Tower Hospital, Huanggang Central Hospital
4
Mitochondrial Protein Analysis by Western Blot
Fibroblasts were lysed in RIPA buffer containing 1 × complete protease inhibitor (Roche). Western blot analysis was performed with antibodies against Miro1 (WH0055288M1; Sigma–Aldrich, Munich, Germany), LC3-I/II (2775; Cell Signaling), Hsp60 (4870; Cell Signaling), Tom20 (sc-17764; Santa Cruz Biotechnologies), Rab9 (sc-74482; Santa Cruz Biotechnologies), MnSOD (ab13533; Abcam), anti-V5 (R960-25; Novex, R96125; Sigma–Aldrich), and β-actin (MA1-744; Thermo Scientific). Mitochondrial fractionation was performed as described previously (12 (link)).
Corresponding organizations : University of Luxembourg, Hertie Institute for Clinical Brain Research, University of Tübingen, University of California, San Diego, University of Lübeck, Helmholtz Zentrum München, Centre Hospitalier de Luxembourg
5
Oxidative Stress and Autophagy Modulation
Glutathione (GSH), N-acetyl cysteine (NAC), hydrogen peroxide (H2O2), sodium pyruvate (Na Py), catalase (Cat), superoxide dismutase (SOD), chloroquine (CQ), 3-methyl adenine (3-MA), Bafilomycin A1, dimethyl sulfoxide (DMSO), Mito Peroxy Yellow 1 (MitoPY1), anti-rabbit IgG, and anti-mouse IgG were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified essential medium (DMEM), Opti MEM medium, phosphate buffered saline (PBS), trypsin-EDTA, and fetal bovine serum (FBS) were bought from GIBCO BRL (Grand Island, NY, USA). Oxidation sensitive 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR, USA). Primary antibodies against α-SMA, Desmin, GFAP, LC3-I/II, ULK1, p-ULK1, mTOR, p-mTOR, Atg5, Atg7, Atg9, Atg14, P62, PI3 Kinase Class III (PI3KCIII), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against α1(I) procollagen was purchased from Epitomics (San Francisco, CA). Primary antibodies against Perilipin A, Rab25, and Fibronectin were purchased from Abcam Technology (Abcam, Cambridge, UK). Atg5 siRNA, Rab25 siRNA, negative control siRNA, Atg5 plasmid constructs and negative control vectors were purchased from Hanbio (Shanghai, China). MegaTran 1.0 transfection reagent was from OriGene (Rockville, MD, USA).
Corresponding organizations : Nanjing University of Chinese Medicine, Saint Louis University
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