Protein extracts from cultured cells were obtained by homogenization in RIPA buffer (Santa Cruz Biotechnology), then cleared by centrifugation at 12,000 rpm for 20 min. For immunodetection, 50 µg total protein from tumor tissue or cultured cells were mixed with Laemmli sample buffer, boiled, separated in 12 or 15% SDS-PAGE, and transferred in a PVDF membrane (Amersham-GE Healthcare). Membranes were incubated overnight using a 1:1,000 (v/v) dilution of the anti-ULK1 (Cell Signaling), 1:3,000 of the anti-Beclin-1 (Cell Signaling), 1:3,000 of the anti-LC3 (Cell Signaling), 1:3,000 of the anti-ATG4b (Santa Cruz), and 1:3,000 of the anti-HIF-1alpha (GeneTex); for detection, 1:2,500 (v/v) dilutions of HRP anti-rabbit or anti-mouse conjugate antibodies (Santa Cruz Biotechnology) were used. Finally, using the Super Signal West Femto chemiluminescent substrate (Thermo Scientific), the membranes were scanned in the C-Digit blot scanner (Li-Cor) and the images were analyzed for densitometry in the associated Image Studio software (LiCor). Membranes were stripped and re-probed for detection of actin (anti-actin, Sc-47778) as a loading control. A representative image from three independent experiments is shown.
Anti lc3
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom, Italy, Japan
Anti-LC3 is a primary antibody that recognizes the microtubule-associated protein 1 light chain 3 (LC3). LC3 is a widely used marker for autophagy, a cellular process involving the degradation and recycling of cellular components. The Anti-LC3 antibody can be used to detect and quantify levels of LC3 in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Anti beclin 1, by Cell Signaling Technology (42 mentions)
Anti p62, by Cell Signaling Technology (38 mentions)
Anti β actin, by Cell Signaling Technology (22 mentions)
Anti caspase 3, by Cell Signaling Technology (19 mentions)
Anti gapdh, by Cell Signaling Technology (19 mentions)
Anti parp, by Cell Signaling Technology (18 mentions)
Anti mtor, by Cell Signaling Technology (16 mentions)
Anti akt, by Cell Signaling Technology (15 mentions)
Anti bax, by Cell Signaling Technology (14 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (13 mentions)
Anti bcl 2, by Cell Signaling Technology (13 mentions)
Anti atg5, by Cell Signaling Technology (13 mentions)
Anti atg7, by Cell Signaling Technology (12 mentions)
Anti ulk1, by Cell Signaling Technology (11 mentions)
Anti β actin, by Merck Group (11 mentions)
Anti p62, by Abcam (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Anti p mtor, by Cell Signaling Technology (9 mentions)
Anti p akt, by Cell Signaling Technology (8 mentions)
Rapamycin, by Merck Group (8 mentions)
225 protocols using anti lc3
1
Western Blot Analysis of Autophagy Markers
2
Western Blot Analysis of LC3, p62, and GAPDH
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WB analysis was performed as previously described, with some modifications [14 (link),15 (link)]. Cell lysates (protein mass was 10 μg) were loaded onto a 4–15% polyacrylamide gradient gel. To analyze LC3 levels, proteins were incubated on PVDF membranes with rabbit polyclonal anti-LC3 (Cell Signaling Technology, Danvers, MA, USA; #12741, 1:1000) as the primary antibody. The PVDF membranes were incubated overnight at 4°C, washed four times, and incubated with a secondary HRP-linked anti-rabbit IgG antibody (Thermo Fisher Scientific Inc., Waltham, MA, USA; #31458, 1:3000) at room temperature for 1 h. Membranes were washed, immunoreactive proteins were detected, followed by SB analysis. The primary and secondary antibodies were removed from the PVDF membranes using WB stripping solution. Membranes were then washed and blotted with rabbit monoclonal [EPR18351] anti-p62 antibody (Abcam, Cambridge, UK; ab207305, 1:80,000), anti-GA-3 phosphate dehydrogenase (GAPDH) antibody (Abcam; ab8243, 1:10,000), HRP-linked anti-rabbit IgG antibody (1:3000), and HRP-linked anti-mouse IgG antibody (#31432,1:5000). To analyze p62 and GAPDH proteins. PVDF membranes were incubated with anti-p62 and anti-GAPDH antibodies at room temperature for 2 h, followed by HRP-linked anti-rabbit IgG antibody and HRP-linked anti-mouse IgG antibody at room temperature for 1 h.
3
MTT and DMSO Assay for Cell Viability
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI medium, Dulbecco’s minimum essential medium (DMEM), fetal bovine serum, horse serum, and Fura-2 AM were obtained from Invitrogen (Carlsbad, CA). Anti-LC3, anti-p62, anti-beclin-1, anti-PI3K, anti-phosphorylated (p)-PI3K, anti-Akt, anti-p-Akt, anti-mTOR, anti-p-mTOR, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). N-Adamantyl-4-methylthiazol-2-amine (KHG26693) was synthesized and characterized as previously described (16 (link)).
4
Monitoring Protein Markers During Cellular Differentiation
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Protein lysates were prepared in a RIPA lysis buffer using ice and a 27 G needle with the addition of protease (Roche, 11836170001, Indianapolis, IN, USA) and phosphatase (Roche 4906837001) inhibitors. Quantification was performed using a detergent-compatible Bradford reagent (Thermo Scientific, 1863028, Waltham, MA, USA). The proteins were loaded into a 12% stain-free gel, and blocking was performed using 3% low-fat milk for 2 h. The membrane was incubated overnight at +4 °C with the following antibodies: anti-PINK1 (Abcam, ab23707, Waltham, MA, USA) 1:1000, anti-LC3 (Cell Signalling, #3868) 1:1000, anti-FUNDC1 (Novus Biologicals, NBP1-81063, Centennial, CO, USA) 1:2500, and anti-p62 1:15,000 (Abcam, ab109012). The membranes were then washed 3 times in TBST and incubated with secondary antibodies (1: 200,000) for 1 h. Subsequent detections were accomplished using the Femto SuperSignal chemiluminescent reagent (Thermo Scientific, 34095). Because of the low protein yield on D1, Western blot was only performed on D7 and D14 of differentiation. The results obtained with the Western blot were normalized to the total protein amount. All images of the Western blots can be found in the supplementary data .
5
Protein Extraction and Western Blot Analysis
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Cells were plated and treated as described in section 2.3 . After 24 h, culture medium was removed, and cells were washed with PBS. To obtain total protein extracts, cell lysis was performed chemically, with Lysis Buffer (5 mM Hepes pH 7.5, 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1.5 mM EGTA, 4 nM PMSF, 1% Aprotinine, 20 nM sodium pyrophosphate, 10 nM sodium orthovanadate), and mechanically, with a cell scarper.
Total protein samples were then clarified by centrifugation (13,000g, 15 min, 4 °C) and quantified using Bradford method.
10 μg of proteins were loaded in a polyacrylamide gel and after SDS-PAGE electrophoresis, western blotting was performed as described by antibodies manufacturers (anti-ERα, 1:1000, Santa Cruz, USA; anti-BRCA1, 1:50, Cell Signaling, USA; anti-CAV1, 1:1000, Cell Signaling, USA; anti-actin, 1:1000, Santa Cruz, USA; anti-LC3, 1:1000, Cell Signaling, USA; anti-Beclin1, 1:1000, Cell Signaling, USA; anti-rabbit, 1:2000, PerkinElmer, USA; anti-mouse, 1:2000, Chemicon, USA; anti-goat, 1:2000, Chemicon, USA).
Total protein samples were then clarified by centrifugation (13,000g, 15 min, 4 °C) and quantified using Bradford method.
10 μg of proteins were loaded in a polyacrylamide gel and after SDS-PAGE electrophoresis, western blotting was performed as described by antibodies manufacturers (anti-ERα, 1:1000, Santa Cruz, USA; anti-BRCA1, 1:50, Cell Signaling, USA; anti-CAV1, 1:1000, Cell Signaling, USA; anti-actin, 1:1000, Santa Cruz, USA; anti-LC3, 1:1000, Cell Signaling, USA; anti-Beclin1, 1:1000, Cell Signaling, USA; anti-rabbit, 1:2000, PerkinElmer, USA; anti-mouse, 1:2000, Chemicon, USA; anti-goat, 1:2000, Chemicon, USA).
6
Thapsigargin and Rapamycin Induced Cell Stress Response
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Thapsigargin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was from Invitrogen (Carlsbad, CA, USA). DMEM and FBS were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The Hoechst kit and Lyso-Tracker Red probe for acidic lysosome staining were from Beyotime (Haimen, Jiangsu, China). Anti-PERK, Anti-BiP, anti-p-eIF2α, anti-eIF2α, anti-cleaved-caspase 3, anti-LC3, anti-p-AKT, anti-p-p70S6K1, anti-p70S6K1, anti-p-4EBP1 and anti-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ATF-6, anti-p62 and anti-GAPDH were from Abcam (Cambridge, UK). The CHOP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were obtained from Sigma-Aldrich with the highest purity available.
7
Western Blot Analysis of Autophagy Proteins
8
Protein Extraction and Quantification
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To monitor protein levels, frozen muscles were pulverized by means of Qiagen TissueLyser and protein extracts were prepared in an appropriate buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10% glycerol, 2% SDS, 1% Triton X-100, Complete EDTA-free protease inhibitor mixture (Roche), 1 mM PMSF, 1 mM NaVO3, 5 mM NaF, and 3 mM β-glycerophosphate. 40 μg of total proteins was loaded, according to BCA quantification. Proteins were separated by SDS-PAGE electrophoresis, in commercial 4-12% acrylamide gels (Thermo Fisher Scientific), and transferred onto nitrocellulose membranes (Thermo Fisher Scientific) by semidry electrophoretic transfer. Blots were blocked for 1 hour at RT with 5% nonfat dry milk (Bio-Rad) in TBS-tween (0.5 M Tris, 1.5 M NaCl, and 0.01% Tween) solution and incubated at 4°C with primary antibodies. Secondary antibodies were incubated 1 hr at RT. The following primary antibodies were used: anti-LC3 (1 : 1000, Cell Signaling), anti-p62 (1 : 5000, Sigma-Aldrich), anti-actin (1 : 20000, Santa Cruz), and anti-MCU (1 : 1000 Sigma-Aldrich). Secondary HRP-conjugated antibodies were purchased from Bio-Rad and used at 1 : 5000 dilution.
9
Protein Extraction and Western Blot Analysis
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Whole cell lysates were prepared from harvested cells using radioimmunoprecipitation (RIPA) buffer (Life Technologies, Warrington, UK) containing Halt phosphatase and protease inhibitors (Thermo Scientific, Altrincham, UK). The total concentration of protein was determined using Peirce BCA Protein Assay Kit (Life Technologies, Warrington, UK) following the manufacturer’s instructions. The following antibodies were used: anti LC3 (Cell Signaling Technology, Beverly, MA, USA), anti-p62 (Cell Signalling Technology), anti-p53 (DO1) (Santa Cruz, Dallas, TX, USA), anti-p27kip1 (Santa Cruz, Dallas, TX, USA), anti-pγH2AX (Cell Signalling Technology) and anti-GAPDH (Origene, Cambridge, UK). Densitometry was performed using UVP imaging software to quantify relative amounts of proteins detected on Western blot membranes. Detailed information can be found in Supplementary Figure S3 .
10
Western Blot Analysis of LC3 and G6PD
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1.5 × 106 cells (2008-C13) were plated in 100 mm cell culture dish and allowed to attach overnight. After 48 hours, cells were lysed with ice-cold lysis buffer supplemented with the protease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany). The protein content was determined by Lowry procedure (Bio-rad DC Protein Assay, MA, USA). Equal amounts of protein (40 μg) were loaded on a polyacrylamide gel and electrophoretically separated in running buffer. After electrophoresis, the proteins were blotted onto an Hybond-P PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). After blocking, the membrane was exposed to the elected primary antibodies: anti-LC3 (1:1000; Cell Signaling, MA, USA) or anti-G6PD (1:500; Santa Cruz Biotechnology, Inc., Europe). After washing, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, USA). The signal was visualized with enhanced chemoluminescent kit (Amersham Biosciences) according to the manufacturer's instructions and analyzed by Molecular Imager VersaDoc MP 4000 (Bio-rad). LC3 and G6PD were normalized to beta-actin (1:7000; AbCam, Cambridge, UK).
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