The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Tris HCl, 20 mM, pH 7.4, NaCl 150 mM, EDTA 4 mM, Triton X-100 1%, and SDS 0.2%) supplemented with phosphatase inhibitors (Na3VO4 1 mM, NaF 1 mM) and phenylmethylsulfonylfluoride (PMSF) 1 mM, purchased from Sigma-Aldrich, and protease inhibitor cocktail (Roche, Penzberg, Germany). The protein lysates were fractionated on SDS-PAGE (10% or 15%), and transferred to a nitrocellulose membrane (Whatman) (GE Healthcare, Little Chalfont, UK). Western blotting was performed using ALK (D5F3 XP, Cell Signaling Technology #3633, Ozyme, Saint Cyr l’Ecole, France), phospho-ALK (Y1507) (Cell Signaling Technology, #14678), LC3-B (Cell Signaling Technology #2775), p62 (BD Transduction Laboratories #610832), ULK1 (Cell Signaling Technology #6439), phospho-ULK1 (Ser757) (Cell Signaling Technology #14202), RAF1 (Cell Signaling Technology #3738), MEK1/2 (Cell Signaling Technology #9122), phospho-MEK1/2 (Ser217/221) (Cell Signaling Technology #9121), ERK1/2 (Cell Signaling Technology #9102), phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology #9106), β-actin (Santa Cruz #7210, Heidelberg, Germany), and HSP90 (Origene #TA332385, Rockville, MD, USA) antibodies. The proteins were visualized using the Clarity™ ECL Western Blotting Substrate (Bio-Rad, Hercules, CA, USA).
Phospho ulk1 ser757
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-ULK1 (Ser757) is a lab equipment product that detects the phosphorylation of ULK1 at serine 757. ULK1 is a key regulator of autophagy, and phosphorylation at Ser757 is involved in the regulation of this process.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (4 mentions)
Phospho p70 s6 kinase thr389, by Cell Signaling Technology (3 mentions)
Flag tag (flag), by Merck Group (3 mentions)
Phospho ulk1 ser555, by Cell Signaling Technology (3 mentions)
Lamp1, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Giantin, by Abcam (2 mentions)
Phospho s6 ser240 244, by Cell Signaling Technology (2 mentions)
Lamp2, by Santa Cruz Biotechnology (2 mentions)
Slc38a9, by Merck Group (2 mentions)
Lamtor1, by Cell Signaling Technology (2 mentions)
Lamtor3, by Cell Signaling Technology (2 mentions)
Atp6v1b2, by Abcam (2 mentions)
Atp6v1a, by GeneTex (2 mentions)
Mouse anti rabbit igg conformation specific, by Cell Signaling Technology (2 mentions)
Lbpa z plbpa echelon, by Tebu-Bio (2 mentions)
Raptor, by Cell Signaling Technology (2 mentions)
Mms 101p, by Fortrea (2 mentions)
Hrp conjugated antibody, by Jackson ImmunoResearch (2 mentions)
17 protocols using phospho ulk1 ser757
1
Immunoblotting of Signaling Proteins
2
Antibody Profiling of Nutrient Sensing Pathways
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Antibodies used were SLC38A9 (HPA043785 Sigma), LAMTOR1 (8975 Cell Signaling), LAMTOR3 (8169 Cell Signaling), RAGA (4357 Cell Signaling), RAGC (5466 Cell Signaling), phospho-p70 S6 Kinase (Thr389) (9234 Cell Signaling), phospho-S6 (Ser240/244) (2215 Cell Signaling), phospho-ULK1 (Ser757) (6888 Cell Signaling), raptor (2280 Cell Signaling), ATP6V1B2 (ab73404 Abcam), ATP6V1A (GTX110815 GeneTex), mouse anti-rabbit IgG (conformation specific) (3678 Cell Signaling), LAMP1 (555798 Pharmingen and ab25630 Abcam), LAMP2 (sc-18822 Santa Cruz), CD63 (H5C6 DSHB), LBPA (Z-PLBPA Echelon, Tebu-bio), EEA1 (sc33585 Santa Cruz), Giantin (ab24586 Abcam), p70 S6 kinase (sc-230 Santa Cruz), ULK1 (8054 Cell Signaling), Tubulin (ab7291 Abcam), RCC1 (sc55559 Santa Cruz), HA (H6533 Sigma, 3724 Cell Signaling, MMS-101P Covance or sc-805 Santa Cruz), V5 (ab9116 Abcam), His (A7058 Sigma), FLAG (F7425 Sigma). The secondary antibodies used were goat anti-mouse AlexaFluor568 (A-11004 and A-11031 Molecular probes), goat anti-rabbit AlexaFluor568 (A-11036 Molecular probes), goat anti-mouse AlexaFluor488 (A-11001 Molecular probes), goat anti-rabbit AlexaFluor488 (A-11008 Molecular probes) and HRP-conjugated antibodies (Jackson ImmunoResearch).
3
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).
4
Western Blot Analysis of Mammary Tissues
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The western blot analysis was conducted as previously described [17 (link)]. The left fourth mammary parenchymal tissues and mammary epithelial cells were lysed with cold lysis buffer (Beyotime), centrifuged at 12000 g for 30 min at 4°C, and the supernatant were mixed with loading buffer (Bio-Rad) and mercaptoethanol and boiled for 5 min. Samples were separated by SDS-PAGE with 10% or 15% acrylamide gel and transferred to PVDF membranes. Primary antibodies were Phospho-p70 S6 Kinase (Thr389) (#9205, 70 kDa, 1 : 1000, Cell Signaling Technology), p70 S6 Kinase (#9202, 70 kDa, 1 : 1000, Cell Signaling Technology), Phospho-4E-BP1 (Thr37/46) (#9459, 15-20 kDa, 1 : 1000, Cell Signaling Technology), 4E-BP1 (#9452, 15-20 kDa, 1 : 1000, Cell Signaling Technology), Phospho-ULK1 (Ser757) (#14202, 140-150 kDa, 1 : 1000, Cell Signaling Technology), ULK1 (#8054, 150 kDa, 1 : 1000, Cell Signaling Technology), Beclin-1 (#3495, 60 kDa, 1 : 1000, Cell Signaling Technology), LC3A/B (#12741, LC3A/B-I: 16 kDa, LC3A/B-II: 14 kDa, 1 : 1000, Cell Signaling Technology), PCNA (#2586, 36 kDa, 1 : 2000, Cell Signaling Technology) and β-actin (#4967, 45 kDa, 1 : 1000, Cell Signaling Technology). The secondary antibodies were purchased from Beyotime. The protein bands were quantified by Image Lab (Bio-Rad).
5
Immunoblotting Analysis of Hippocampal Proteins
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The protein lysates from the hippocampi of WT or Trpm2−/− mice were prepared in a RIPA cell lysis buffer (GenDEPOT), containing a protease inhibitor cocktail (Roche). These lysates were then subjected to an 8% SDS-PAGE gel. The proteins were transferred to PVDF membranes and then treated for 1 h with a TBS-T solution (20 mM of Tris/HCl, 500 mM of NaCl, 0.1% Tween 20), containing 2–5% skimmed milk powder. They were incubated with primary antibodies against β-actin (Sigma), α-tubulin (Millipore), GAPDH (Santa Cruz), calnexin (Santa Cruz), EEA-1 (Abcam), LAMP-1 (Santa Cruz), or autophagy-related proteins (LC3B:#2755, mTOR: #2972, phospho-mTOR: #5536, Raptor:#2280, AMPKα: #2532, phospho-AMPKα: #2531, AMPKβ1: #12063, phospho-AMPKβ1: #4181, ULK1: #8054, phospho-ULK1 (Ser555): #5869, phospho-ULK1 (Ser757): #14202, from Cell Signaling) at 4 °C on a rotary shaker overnight. The membranes were washed three times in a TBS-T solution, incubated with a secondary antibody for 1 h, and then treated with WEST-ZOL® ECL solution (iNtRON biotech). Blots were analyzed using an ImageQuant™ LAS 4000 chemiluminescence (GE Healthcare).
6
Western Blotting Analyses of Autophagy Pathway Proteins
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Total cell extracts were prepared and assayed by western blot as previously described [36 (link)–38 (link)]. The following primary antibodies and dilutions were used: ULK1 (Cell Signaling Technology, 8054, 1:2000), Phospho-ULK1 (Ser757) (Cell Signaling Technology, 14202, 1:2000), Phospho-ULK1 (Ser317) (Cell Signaling Technology, 37762, 1:2000), Phospho-ULK1 (Ser555) (Cell Signaling Technology, 5869, 1:2000), LC3B (SellckChem, A5202, 1:2000), mTOR (Santa Cruz Biotechnology, sc-517464, 1:2000), P62/SQSTM1 (SellckChem, A5180, 1:2000), P53 (Santa Cruz Biotechnology, sc-47698, 1:2000), p-P53 (Ser315) (WanLeiBio, WLP1333, 1:2000), and GAPDH (Santa Cruz Biotechnology, sc-25778,1:5000). The following secondary antibodies were also used: anti-mouse IgG-horseradish peroxidase (HRP), anti-rabbit IgG-HRP, and anti-goat IgG-HRP (Santa Cruz Biotechnology). Bound antibodies were visualized with the Luminata Forte Western HRP substrate (Millipore).
7
Autophagy Pathway Protein Analysis
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The following primary antibodies were used: pan-LC3 (MBL, #PM036); LAMP1 (Santa-Cruz, sc-17768); phospho-Ulk1 Ser757 (Cell Signaling, #6888S); phospho-Ulk1 Ser555 (Cell Signalling, #5869); Tom20 (Santa-Cruz, sc-17764); phospho-AMPK T172 (Cell Signaling, #2535S); phospho-4E-BP1 Thr37/46 (Cell Signaling, #2855S); phospho-42/44 MAPK Thr202/Tyr204 (Cell Signaling, #4377S); mTOR (Cell Signaling, #2983S); pTSC2 (Cell Signalling, #3617), GAPDH (Cell signaling, #2118), Tubulin (Sigma, T5168). The following secondary antibodies were used: Goat-anti-Rabbit IgG (H+L) Alexa Flour 488 (Invitrogen, A11088); Rabbit-anti-Mouse IgG (H+L) Alexa Fluor 568 (Invitrogen, A11031); Goat-anti-Rabbit IgG HRP Conjugated (Sigma-Aldrich, A0545); Rabbit-anti-Mouse IgG HRP Conjugated (Sigma-Aldrich, A9044).
8
Quantitative Western Blot Analysis of Macrophage Proteins
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Macrophages stimulated as described above were lysed by lysis buffer. The concentration of protein was determined by the BCA Protein Assay Kit (ZJ102, Epizyme Biotech, Shanghai, China). Appropriate gels (7.5%, 10%, 12.5%, PG211, PG212, PG213, Epizyme Biotech, Shanghai, China) were used in this experiment. After electrophoresis, proteins were transferred to PVDF membranes (Millipore, Burlington, MA, USA) and incubated in 5% skim milk at room temperature for 1 h. PVDF membranes were then incubated in primary antibody against GSDMD (#39754), N-GSDMD (#39754), pro-IL-1β (#12242), p-mTOR (#5536), phospho-ULK1 (Ser757) (#14202), p-S6 (#4858), p-4EBP1 (#2855), mTOR (#2983), Raptor (#2280), LC3B (#58139), p62 (#5114), and β-actin (#4970) (1:1000, all from Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After being washed with TBST 3 times, membranes were incubated with corresponding secondary antibodies (1:5000, ab6721, Abcam, Cambridge, UK) for 2 h. The ECL kit (SQ201, Epizyme Biotech, Shanghai, China) was used to detect the protein in the membrane and ImageJ software (Version 1.54b) was used for protein quantification. Three replicates were used in Western blot.
9
Antibody Profiling of Nutrient Sensing Pathways
10
Western Blot Analysis of Autophagy Markers
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Lysates were prepared from tumor cells and analyzed by western blotting. Primary antibodies include FIP200 (1:1000, Cell Signaling #12436), p62 (1:2000, Cell Signaling #39749), TSC1 (1:1000, Cell Signaling #6935), phospho-ULK1 (Ser757) (1:1000, Cell Signaling #6888), ULK1 (1:1000, Cell Signaling #8054), LC3B (1:2000, Cell Signaling #43566), phospho-p70 S6K (Thr389) (1:1000, Cell Signaling #9205), p70 S6K (1:1000, Santa Cruz #sc-230), Atg5 (1:1000, Cell Signaling #12994), Atg7 (1:1000, Cell Signaling #8558), phospho-Stat3 Y705 (1:1000, Cell Signaling #9145), Stat3 (1:1000, Cell Signaling #3139), Osteopontin (1:1000, R&D Systems #MAB808), GAPDH (1:5000, Cell Signaling #5174), β-actin (1:10000, Sigma-Aldrich #), Vinculin (1:10000, Sigma-Aldrich #V4505) and HRP-linked anti-Rabbit IgG or anti-Mouse IgG (1:10000, Cell Signaling) were used as secondary antibodies. ECL HRP substrate (Thermo Scientific) was used for signal imaging.
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