Nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo‐Fisher). The following primary antibodies were used for western blot analysis: anti–phosphorylated c‐Jun NH2‐terminal kinase (P‐JNK; no. 9255; Cell Signaling Technology), anti‐JNK (no. 9525; Cell Signaling Technology), anti–phosphorylated extracellular signal–regulated kinase (P‐Erk) 1/2 (no. 4376; Cell Signaling Technology), anti‐Erk1/2 (no. 5013; Cell Signaling Technology), anti‐P‐p38 (no. 9216; Cell Signaling Technology), anti‐p38 (no. 9212; Cell Signaling Technology), anti‐LC3I/II (no. 4108; Cell Signaling Technology), anti‐Nrf2 (no. 137550; Abcam), anti‐p62 (no. 109012; Abcam), and anti‐Slc7a11 (no. 37185; Abcam).
Anti lc3 1 2
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-LC3 I/II is a primary antibody that recognizes the microtubule-associated protein 1A/1B-light chain 3 (LC3), a marker for autophagy. This antibody detects both the cytosolic (LC3-I) and lipidated (LC3-II) forms of LC3, which are essential for the formation of autophagosomes.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Cell Signaling Technology (8 mentions)
Anti beclin 1, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti p62, by Abcam (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti p62, by Cell Signaling Technology (3 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti mtor, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti nrf2, by Abcam (2 mentions)
Anti parkin, by Cell Signaling Technology (2 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti bcl 2, by Cell Signaling Technology (2 mentions)
Anti sqstm1, by Cell Signaling Technology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Anti atg5, by Cell Signaling Technology (2 mentions)
Anti pi3k, by Cell Signaling Technology (2 mentions)
Anti beclin1, by Abcam (2 mentions)
22 protocols using anti lc3 1 2
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting Analysis of Autophagy Markers
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The following primary antibodies were used in this study: anti-PIWIL1 antibody (ab12337; Abcam, Cambridge, UK), anti-p-mTOR antibody (ab109268; Abcam), anti-LC3 I/II (#2775S; Cell Signaling Technology, Danvers, MA, USA), anti-P62/SQSTM1 (#8025S; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-phosphor-NAK/TBK (ab109272; Abcam), anti-NAK/TBK (A5497; Bimake), anti-optineurin (ab213556; Abcam), anti-β-actin (#4970; Cell Signaling Technology), anti-Akt (#4691; Cell Signaling Technology), anti-phospho-Akt (Ser473) (#9271; Cell Signaling Technology), anti-Ki67 (#4685; Cell Signaling Technology), anti-Bcl-2 (A5010; Bimake), anti-OCT4 (60242-1-Ig; Proteintech), and anti-Nanog (#4903T; Cell Signaling Technology). Goat anti-mouse and goat anti-rabbit antibodies (Biosciences) were used as the secondary antibodies for western blotting. Dexamethasone and doxorubicin were purchased from Sigma (St. Louis, MO, USA), and bortezomib was purchased from Selleck Chemicals. 3-Methyladenine (3-MA) and the mitophagy inhibitor cyclosporin A (CsA) were purchased from MedChemExpress (NJ, USA). All were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C for up to 6 months. For all cell-based experiments, drugs were diluted at least by 1:1000 to ensure that the final DMSO concentration was lower than 0.1%.
3
Western Blot Analysis of Cell Signaling
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Proteins were extracted from cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Total proteins were separated by SDS-PAGE gel and then transferred into PVDF membranes (Millipore). After incubating with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies (Cell Signaling Technology, Beverly, MA., USA) used in this study include anti-Beclin-1 (No. 4122, 1:1000), anti-LC3 I/II (No.12741, 1:1000), anti-p62 (No.88588, 1:1000), anti-FOXM1 (No.5436, 1:1000), anti-p-AKT (No.4060, 1:2000), anti-mTOR (No.2983, 1:1000) and anti-GAPDH (No.5174, 1:2500). Then, the membranes were incubated with corresponding secondary antibodies (Cell Signaling Technology, No.7076 and NO.7074, 1:4000) at room temperature for 1 h. ECL system (Bio-Rad Laboratories) was used for detection of antibody-bound proteins according to manufacturer's instructions.
4
Western Blot Analysis of Stem Cell Markers
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Total proteins were extracted using RIPA lysis buffer on ice, electrophoresed on 12% SDS-PAGE gels (Bio-Rad) and blotted onto nitrocellulose membranes (Amersham Biosciences Corp, Sunnyvale, CA). The membranes were blocked with 5% non-fat milk powder at room temperature for 2 hours and incubated overnight with primary antibodies: anti-ATG4A (1:400) (Abcam, Cambridge, UK), anti-E-cadherin (1:2000) (BD Transduction Laboratories, Franklin Lakes, NJ), anti-N-cadherin (1:1000) (BD Transduction Laboratories), anti-vimentin (1:1000) (BD Transduction Laboratories), anti-Sox2 (1:500) (Abcam), anti-Oct4 (1:400) (Abcam), anti-Bmi-1 (1:400) (Abcam), anti-LC3I/II (1:1000) (Cell Signaling Technology, Danvers, MA) and anti-β-actin antibody (1: 2000) (Sigma-Aldrich). After three 5-min washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 2000) (Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature and then washed again in TBS-T and visualized with an enhanced chemiluminescence kit (ECL-kit) (Santa Cruz Biotechnology). All of the experiments were performed in triplicate.
5
Apoptosis Signaling Proteins in Heart Tissue
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The expression levels of Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, LC3I/II, beclin-1, p62, and β-actin were measured by western blot. The proteins in each sample were extracted from heart tissue, and the protein concentration was determined with a BCA protein assay. Then, 40 μg of protein from each sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked in 5% fat-free milk and incubated with the appropriate primary antibodies overnight followed by incubation with secondary antibodies. Then, the bands were detected using an enhanced chemiluminescence (PerkinElmer, Waltham, MA, United States) kit and quantified by densitometry using AlphaEaseFC software. The primary antibodies were as follows: anti-p62, anti-Bax (1:1,000; Abcam, Cambridge, United Kingdom); anti-Bcl-2, anti-LC3I/II, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-interleukin (IL-)-1, anti-tumor necrosis factor (TNF-)-α, anti-GAPDH (1:1,000; Cell Signaling Technology, Danvers, MA, United States); and anti-β-actin (1:5,000; Bioword, Shanghai, China).
6
Liver Tissue Histopathology and Mitochondrial Analysis
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Fresh liver tissues were fixed in 10% formaldehyde, dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). H&E staining of livers was assessed under an optical microscope (Zeiss, Thuringia Germany). Liver injury scores were evaluated in 8 randomly selected, nonoverlapping fields at 200× magnification in each section. The sections were analyzed by two professional pathologists who were blinded to the experimental protocol and scored the liver damage.
To visualize the mitochondria, cells were cultured on cover slips and then incubated using a MitoTracker Red fluorescent probe kit (300 nM at 37°C for 30 minutes; Invitrogen, Waltham, USA). For immunostaining, the cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 at RT. Cells were incubated with the specific primary antibody anti-LC3-II/I (12741, Cell Signaling) at 4°C overnight followed by incubation with a fluorescent secondary antibody. After counterstaining with 4’ 6-diamidino-2-phenylindole (DAPI), the slides were observed using a confocal inverted laser microscope (Zeiss, Germany).
To visualize the mitochondria, cells were cultured on cover slips and then incubated using a MitoTracker Red fluorescent probe kit (300 nM at 37°C for 30 minutes; Invitrogen, Waltham, USA). For immunostaining, the cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 at RT. Cells were incubated with the specific primary antibody anti-LC3-II/I (12741, Cell Signaling) at 4°C overnight followed by incubation with a fluorescent secondary antibody. After counterstaining with 4’ 6-diamidino-2-phenylindole (DAPI), the slides were observed using a confocal inverted laser microscope (Zeiss, Germany).
7
Nectin-4-MMAE Autophagy Modulation and Apoptosis Assay
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Nectin-4-MMAE is a gift from Mabwell Bioscience CO., LTD. Shanghai China. Autophagy modulators: LY294002 (Medchemexpress, HY-10108, Shanghai China); Chloroquine (Sigma-Aldrich, C6628, Darmstadt Germany); Rapamycin (Sangon Biotech, A606203, Shanghai China). Cell viability assay: MTT (Beyotime, C0009S, Shanghai, China). ADC labeling: AlexaFlour 488 protein label kit (ThermoFisher, A10235, MA US). Cell apoptosis assay: cell apoptosis detection kit (Meilunbio MA0220-1, Dalian China). Autophagy assay: Cyto-ID autophagy detection kit (Enzo Life Sciences, ENZO-51031-K200, NY US); Lyso-Tracker-DND 99 (Invitrogen, L7528, CA US). Antibodies: Primary antibody: anti-GAPDH, #2118; anti-LC3 I/II, #3868; anti-SQSTM1, #8025; anti-caspase 3, #9662; anti-PARP, #9542; anti-phospho-mTOR (Ser2448), #2971; anti-mTOR, #2983; anti-phospho-AKT (Ser473), #4060; anti-phospho-p70s6 Kinase (Ser371), #9208; anti-p70s6 Kinase, #2708; anti-phospho-4EBP1 (Thr45), #2971; anti-4EBP1, #9644, anti-PI3 Kinase, #4257; anti-phospho-PI3 Kinase, #4228 (Cell Signaling Technology, MA US). Anti-PDK-1, AF7707 (Beyotime Biotechnology, Shanghai, China). Anti-PTEN, A19104 (Abclonal, Wuhan China). Secondary antibody: HRP-conjugated anti-rabbit secondary antibody, #7074; anti-mouse IgG secondary antibody, #7076 (Cell Signaling Technology, MA US).
8
Exploring Autophagy in Bone Metabolism
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β-GP (50020), 17-β estrogen (E8875), Rapamycin (37094), 3MA (M9281) and Fulvestrant (V900926) were from Sigma-Aldrich. Methy-piperidinopyrazole (MPP, ERα selective antagonist) and 2-phenyl-3-(4-hydroxyphenyl)-5, 7-bis (trifluoromethyl)-pyrazolo [1,5-alpha] pyrimidine (PHTPP, ERβ selective antagonist) were from Tocris, and anti-LC3I/II was from Cell Signaling. Anti-β-actin was from Santa Cruz Biotechnology, and anti-Runx2 and anti-ATG5 were from Abgent. The GFP–LC3 expression vector was from Origene and Lipofectamine 2000 was from Invitrogen. siRNA against mouse Atg5 was synthesized by GeneChem. The ALP kit was from Jiancheng Nanjing Biological Engineering, and Alexa Fluor R488 donkey anti-rabbit was from Abcam. Dulbecco’s Modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were from Gibco-BRL, and RIPA lysate was from Beyotime.
9
Western Blotting of RAGE and STAT3 Signaling
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Western blotting was performed as previously described [22 (link)]. Anti-RAGE (1:1000, #6996S; Cell Signaling Technology, Inc., MA, USA), anti-STAT3 (1:1000, #12640; Cell Signaling Technology, Inc., MA, USA), anti-phosphorylated STAT3 (1:2000, #9145; Cell Signaling Technology, Inc., MA, USA), anti-cleaved caspase 3 (1:1000, #9664; Cell Signaling Technology, Inc., MA, USA), anti-LC3II/I (1:1000, #4108; Cell Signaling Technology, Inc., MA, USA), anti-Beclin1 (1:1000, #3495; Cell Signaling Technology, Inc., MA, USA) and anti-GADPH (1:1000, #2118; Cell Signaling Technology, Inc., MA, USA) were used as primary antibodies. The membranes were incubated with primary antibodies at 4℃ overnight, followed by incubation with HRP‐conjugated secondary antirabbit antibody (1:50,000; cat. no. BM2006; BOSTER Biological Technology Co., Ltd., Wuhan, Hubei, China) at 37 °C for 1 h after washing. anti-GADPH was used as an endogenous control for other proteins. Images were obtained using a chemiluminescent western blot scanner.
10
Western Blot Analysis of FcRn, ATG5, and LC3
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Cells were washed with PBS and lysed in Laemmli buffer (Invitrogen, USA) containing 50 mM DTT. Lysates from 2x105 cells were analysed in singlicate by 15% SDS-PAGE, and proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in PBS and incubated with the antibodies anti-FcRn (Santa Cruz Biotechnology, USA sc-271745), anti-ATG5 (Cell Signaling Technology, USA, #12994) and anti-LC3-I/II (Cell Signaling Technology, #4108S). Anti-β-actin (Santa Cruz Biotechnology, #sc-47778) or anti-GAPDH (Santa Cruz Biotechnology, # sc-365062) was a loading control. The membranes were developed with an enhanced chemiluminescence Western blot detection reagent (Thermo Scientific, USA). Uncropped Western blot and replicates are presented as Supplemental Figure S1 and S2
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