Cells were placed on ice, washed twice with cold PBS and lysed with NETN buffer [50 mM Tris-HCI (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40] plus protease inhibitors. 30–50 μg protein of each sample was used for analysis by immunoblotting. Quantification of western blot films was performed with Image J, with the measure of controls set as 1. Antibodies used include: BRUCE (Bethyl, A300-367A), LC3B (Cell signaling, 2775 and 3868), phospho-ULK1 (Cell signaling, 5869), phospho-AMPKα (Cell signaling, 2531), ULK1 (Cell signaling, 8054), AMPKα (Cell signaling, 5832), GFP (Santa Cruz, sc-9996), FLAG (Sigma, A8592), GAPDH (Cell signaling, 5174), β-Actin (Cell signaling, 3700), α-Tubulin (Sigma, T9026).
Phospho ulk1
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-ULK1 is a lab equipment product that detects and quantifies phosphorylation of the ULK1 protein. ULK1 is a serine/threonine protein kinase that plays a key role in autophagy regulation. The product can be used to investigate the phosphorylation status of ULK1 in various experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Phospho ampkα, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Ampkα, by Cell Signaling Technology (3 mentions)
Cleaved parp, by Cell Signaling Technology (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Beclin 1, by Cell Signaling Technology (3 mentions)
Phospho ampk, by Cell Signaling Technology (3 mentions)
Phospho acc, by Cell Signaling Technology (3 mentions)
Phospho s6, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Phospho ulk1 ser555, by Cell Signaling Technology (2 mentions)
Total ulk1, by Cell Signaling Technology (2 mentions)
Slc7a11, by Cell Signaling Technology (2 mentions)
Phospho s6k, by Cell Signaling Technology (2 mentions)
Vinculin, by Merck Group (2 mentions)
Ampkα1, by Cell Signaling Technology (2 mentions)
Ampkα2, by Cell Signaling Technology (2 mentions)
Acsl4, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
18 protocols using phospho ulk1
1
Immunoblotting Analysis of Autophagy Signaling
2
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).
3
Immunoblotting for Autophagy Regulators
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Protein extracts were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and phosphatase inhibitor cocktail 2, separated by SDS-PAGE, and then electrotransferred onto polyvinylidene difluoride membranes. Blots were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) (0.5 M, pH 7.4) with 0.2% Tween 20 for 1 h at room temperature and subsequently incubated with primary antibodies at 4°C overnight. The following primary antibodies were obtained from Cell Signaling Technology: Beclin-1 (1:1,000), phospho-Beclin-1 (Ser15) (1:1,000), phospho-Beclin-1 (Ser91/Ser94) (1:1,000), ULK1, phospho-ULK1 (Ser555), phospho-ULK1 (Ser757), AMPKα, phospho-AMPK (Thr172), mTOR, phospho-mTOR (Ser2448), LC3B, SQSTM1/p62, DYKDDDDK tag, MYC tag, and GFP tag. Antibodies recognizing GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and beta-actin were kind gifts from Cell Signaling Technology. Mouse anti-PHEV-N monoclonal antibody (MAb) was provided by our research support group. Subsequently, blots were washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000) for 1 h at room temperature. The immunoreactive bands were visualized using ECL detection reagent (Millipore, Billerica, USA) quantified by ImageJ software.
4
Genotyping and Immunoblotting Techniques
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Primers for genotyping were obtained from Integrated DNA Technologies. Total-CaMKII (ab22609), TBX3 (ab154828), and MLCA2 (MYL-7; ab68086) antibodies were obtained from Abcam. Ox-CaMKII (GTX36254) was purchased from GeneTex. Beta-actin (#8457), LC3 I & II (#12741), phospho-ULK1 (#12753), and total-ULK1 (#6439) were purchased from Cell Signaling Technology. D-Tat-Beclin-1 was obtained from Calbiochem. Pioglitazone (#E6910) and Angiotensin II (#A9525) were purchased from Sigma Aldrich. The enhanced chemiluminescence detection kit was purchased from Advansta.
5
Western Blot Analysis of Autophagy Markers
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Equal amounts of protein were added to loading buffer (BioRad, Hercules, CA, USA) containing 50 μL/mL β-mercaptoethanol and heated to 98°C for 5 minutes and then loaded onto a 4–20% gradient SDS-polyacrylamide gel (BioRad) and run for 60 minutes at 130 volts. The protein was transferred to a nitrocellulose membrane over 1 hour at 400 mA and then blocked in 5% non-fat milk for 1 hour before incubation with primary antibodies. Antibodies against LC3, p62, p-p70S6K, p70S6K, pS6, S6, phospho-AMPK, AMPK, phospho-ULK1, and ULK1 (Cell Signaling, Danvers, MA, USA), PFKFB3 (Proteintech, Chicago, IL, USA), and β-actin (Sigma) were diluted 1:1,000 and incubated overnight at 4°C, with the exception of p62 and β-actin Ab, which were incubated at room temperature for 1 hour. Membranes were washed for 30 minutes in Tris-buffered saline with Tween 20 (TBS-T) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) before addition of secondary antibodies (anti-mouse or anti-rabbit), diluted 1:10,000 in TBS-T (Sigma). ECL Western Blotting Detection Kit (Amersham/GE Pittsburgh, PA, USA) was used to develop membranes. Quantitative densitometry was performed using Image J (NIH).
6
Western Blot Antibody Panel for Cell Signaling
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Primary antibodies against GFP (Takara Bio, 632380, AB_10013427; 1:5000), G-6-PDH (Sigma-Aldrich, A9521, AB_258454; 1:5000), Adh1 (Millipore, 126745, AB_564196; 1:200000), RPS6 (Cell Signaling Technology, 2217, AB_331355; 1:1000), phospho-RPS6 (Ser235-236) (Cell Signaling Technology, 4856, AB_2181037; 1:1000), RPS6KB (Cell Signaling Technology, 2708, AB_390722; 1:1000), phospho-RPS6KB (Thr389) (Cell Signaling Technology, 9205, AB_330944; 1:1000), EIF4EBP1 (Cell Signaling Technology, 9452, AB_331692; 1:1000), phospho-EIF4EBP1 (Thr37/46) (Cell Signaling Technology, 2855, AB_560835; 1:1000), phospho-EIF4EBP1 (Ser65) (Cell Signaling Technology, 9451, AB_330947; 1:1000), AKT1 (Cell Signaling Technology, 4691, AB_915783; 1:1000), phospho-AKT(Ser473) (Cell Signaling Technology, 4060, AB_2315049; 1:1000), phospho-AKT(Thr308) (Cell Signaling Technology, 13038, AB_2629447; 1:1000), ULK1 (Cell Signaling Technology, 8359, AB_11178668; 1:1000), phospho-ULK1(Ser757) (Cell Signaling Technology, 6888, AB_10829226; 1:1000), MAP1LC3 AB (Cell Signaling Technology, 12741, AB_2617131; 1:1000), β-actin (Cell Signaling Technology, 4967, AB_330288; 1:1000) were used.
7
Western Blot Analysis of Liver Proteins
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For western blot analysis, about 10 mg of liver tissue was measured and mixed with 500-µL lysis buffer. The resultant was homogenized and centrifuged at 10000 rpm for 10 minutes, followed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (10–15%). The isolated proteins were transferred to polyvinylidene fluoride membrane, and the blots were then exposed to primary antibodies against Caspase-3/-9/-8, Phospho-ULK1, sXBP1, CHOP, IRE1, LC3 (Cell Signal Technology, Beverly, CA, USA), COL4A1 (GeneTex, Irvine, CA, USA), SQSTM1/P62, and GAPDH (Abcam). The immuno-blots were washed with mixture of tris-buffered saline in tween-20, and were incubated with horseradish peroxidase (HRP) coupled with ant-rabbit IGG antibodies (1:2000), HRP-anti-mouse IGG antibodies (1:1000) and HRP-ant-goat IGG (1:1000) were stored at room temperature for 1 hour. The blots were developed using enhanced chemiluminescence, followed by exposure to film and densitometry analysis.
8
Regulation of MAPK and Autophagy Signaling
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Experiments were performed as previously described [22 (link)]. Antibodies specific for ERK (#4695), JNK (#9252), p38 (#9212), phospho-ERK (Thr202/Tyr204) (#4370), phospho-JNK (Thr183/Tyr185) (#4668), phospho-p38 (Thr180/Tyr182) (#9211), LC3B (#2775S), mTOR (#2983), phospho-mTOR (Ser2448) (#2971), ULK1 (#8054), phospho-ULK1 (Ser555) (#5869), phospho-ULK1 (Ser757) (#14202), phospho-70S6 kinase (Thr389) (#9234), 70S6 kinase(#2708), phospho-4E-BP1 (Thr37/46) (#2855), 4E-BP1 (#9452), AMPK (#2603) and phospho-AMPK (Thr172) (#2531) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against DUSP5 (#ab200708) were from Abcam (Cambridge, MA) and antibodies against p62 (#PM045) were from MBL Life science. Specific inhibitors U0126 (#HY-12031A), SB203580 (#HY-10256), SP600125 (#HY-12041) and bafilomycin A1 (#HY-100558) were obtained from MCE. Rapamycin (#V900930-1MG) and rose bengal (#330000) were from Sigma. U0216 (10 μM, 24 h), SB203580 (20 μM, 24 h), SP600125 (15 μM, 24 h), bafilomycin A1 (25 nM, 6 h) and rapamycin (100 nM, 12 h) were added to the cell cultures as described in different experiments. The primers for DUSP5 were TGTCGTCCTCACCTCGCTA and GGGCTCTCTCACTCTCAATCTTC.
9
Autophagy Protein Expression Analysis
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The Antibodies used in this study were ATG3, ATG4B, ATG7, BECN1, BNIP3, Cathepsin D, LC3-I/II, mTOR, phospho-mTOR (Ser2448), AMPK, phospho-AMPK, phospho-ULK1 (Ser317), Rubicon, phospho-ULK1 (Ser757) (Cell Signaling Technology, Beverly, MA), LAMP-2 (Invitrogen, Carlsbad, CA), TFEB (Novus Biologicals, Littleton, CO), FIP200 (MilliporeSigma, St. Louis, MO), p62 (BD Biosciences, Franklin Lakes, NJ) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). All other reagents used were of analytical grade and purchased from MilliporeSigma (St. Louis, MO).
10
Quantitative Western Blot Analysis of Protein Expression
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Western blotting to analyze protein expression was performed as previously described62 –64 (link). Briefly, cell pellets and tissues were lysed using RIPA lysis buffer (Millipore) and the protein concentration was determined by a Bradford assay (Bio-Rad) using a Nanodrop2000 (Thermo scientific). 15–20 μg of protein was used for immunoblot analysis using antibodies against phospho-AMPKα (Thr172, 1:1000, 2535, Cell Signaling), AMPKα (1:1000, 5832, Cell Signaling) AMPK α1 (1:1000, 2795, Cell Signaling), AMPKα2 (1:1000, 2757, Cell Signaling), phospho-ACC (S79, 3661, 1:1000, Cell Signaling), ACC (1:1000, 3662, Cell Signaling), phospho-S6 (Ser240/244, 1:5000, 3661, Cell Signaling), S6 (1:1000, 2217, Cell Signaling), phospho-S6K (Thr389, 1:1000, 9205, Cell Signaling), S6K (1:1000, sc-230, Santa Cruz), phospho-ULK1 (S757, 6888, 1:1000, Cell Signaling), ULK1 (1:1000, 8054, Cell Signaling), LKB1 (1:1000, sc-32245, Santa Cruz), SLC7A11 (1:3000, 12691, Cell Signaling), GPX4 (1:1000, MAB5457, R&D systems), ACSL4 (1:1000, sc-271800, Santa Cruz), Cleaved Caspase-3 (Asp175, 1:500, 9661, Cell Signaling), Cleaved-PARP (Asp214, 1:1000, 9544, Cell Signaling), Vinculin (1:50000, V4505, Sigma).
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