Antibodies from Cell Signaling Technology are as follows: AMPKα (1:1000; #2532), p-AMPKThr172 (1:1000; #2531), ATG13 (1:1000; #13468), Crkl (1:500; #3182), p-CrklTyr207 (1:100; #3181), mTOR (1:500; #2983), p-mTORSer2448 (1:500; #2971), RPS6 (1:1000; #2317), p-RPS6Ser240/244 (1:1000; #5364), ULK1 (1:1000; #8054), p-ULK1Ser757 (1:500; #6888), p-ULK1Ser555 (1:500; #5869), p-ATG13Ser318/ATGSer355 (1:1000; #46329), LC3B (1:500; #2775), ATG7 (1:1000; #8558), glyceraldehyde phosphate dehydrogenase (GAPDH) (1:1000; #5174), β-tubulin (1:1000; #2146), mouse immunoglobulin G (IgG) (1:5000; #7076), and rabbit IgG (1:5000; #7074). OXPHOS cocktail (1:2000; Abcam, #110413), green fluorescent protein (1:1000; Roche, #118144600001), MT-CO2 (1:2000; Thermo Fisher Scientific, #A-6404), p62 (1:1000; BD, #610832), and p-ATG13Ser318 (1:1000; Abnova, #NBP2-19127) were also used.
P ulk1 ser757
Manufactured by Cell Signaling Technology
Sourced in United States
P-ULK1 (Ser757) is a specific antibody that recognizes the phosphorylated form of ULK1 (Unc-51 Like Autophagy Activating Kinase 1) at serine 757. ULK1 is a key regulator of the initiation of autophagy, a cellular process involved in the degradation and recycling of damaged organelles and proteins.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
P ampk thr172, by Cell Signaling Technology (2 mentions)
P mtor ser2448, by Cell Signaling Technology (2 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Atg13, by Cell Signaling Technology (1 mentions)
P crkltyr207, by Cell Signaling Technology (1 mentions)
P rps6ser240 244, by Cell Signaling Technology (1 mentions)
P ulk1ser555, by Cell Signaling Technology (1 mentions)
Oxphos cocktail, by Abcam (1 mentions)
Mt co2, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Anti rabbit 700, by LI COR (1 mentions)
β actin, by Bioworld Technology (1 mentions)
P p65 ser536, by Abcam (1 mentions)
Hsc70, by Proteintech (1 mentions)
Ring1b, by Cell Signaling Technology (1 mentions)
Gata4, by Proteintech (1 mentions)
Ang 2, by Novus Biologicals (1 mentions)
13 protocols using p ulk1 ser757
1
Comprehensive Western Blot Protocol
2
Quantifying Muscle Protein Signaling
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Quadriceps muscle was homogenized as whole tissue (~30 mg) and mitochondrial isolations (~80 mg) for immunoblotting as previously described (Newsom et al. 2017 ). Approximately 35 μg of protein from tissue or cell lysates was resolved on 7–12% Bis‐Tris gels and transferred to nitrocellulose membranes. Two control samples (20 μg of insulin‐stimulated L6 cell samples) were loaded onto each gel and their average intensity was used to normalize intensities between gels. Ponceau staining of membranes was used to verify equal loading and transfer of protein to the nitrocellulose membrane. Images were generated using infrared detection (Odyssey, Licor, Lincoln NE). Primary antibodies included Akt (no. 2920; Cell Signaling Technology), pAktSer473 (no. 9271; Cell Signaling Technology), LC3 (no. 12741; Cell Signaling Technology), p62 (no. 7695; Cell Signaling Technology), mTOR (no. 4517; Cell Signaling Technology), pmTORSer2448 (no. 2971; Cell Signaling Technology), pUlk1Ser757 (no. 6888; Cell Signaling Technology), AMPK (no. 5832; Cell Signaling Technology), pAMPKThr172(no. 2535; Cell Signaling Technology). Secondary antibodies included anti‐rabbit‐700 (1:10,000; no. 926‐68071; Licor) and anti‐mouse‐800 (1:50,000; no. 926‐32212; Licor).
3
Comprehensive Protein Analysis in Cardiac Cells
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Primary antibodies were against p19 (NB200‐106, Novus Biologicals, CO, USA), p21 (#2947, Cell Signaling Technology, Beverly, MA, USA), p53 (#2424, Cell Signaling Technology, USA), p16 (ab211542, Abcam, Cambridge, MA, USA), renin (#5250, Cell Signaling Technology, USA), Ang II (NBP1‐31127, Novus Biologicals, USA), ANP (#AB5490, Millipore, MA, USA; sc‐515701, Santa Cruz Biotechnology Inc., Dallas, TX, USA, USA), BNP (#DF6902, Affinity Biosciences, OH, USA; ab19645, Abcam, USA), GATA4 (#19530, Proteintech, IL, USA; sc‐25310, Santa Cruz Biotechnology Inc., USA), LC3B (#NB600‐1384, Novus Biologicals, USA), p62 (#39749, Cell Signaling Technology, USA), Bmi‐1 (#5856, Cell Signaling Technology, USA; 66161‐1‐Ig, Proteintech, USA), RING1B (#5694, Cell Signaling Technology), NF‐κB‐p65 (sc‐8008, Santa Cruz Biotechnology Inc., USA; #8242, Cell Signaling Technology, USA), p‐p65 (Ser536) (ab76302, Abcam, USA), IκB‐α (AF1282, Beyotime Biotechnology, Shanghai, China), p‐IκB‐α (Ser32) (sc‐8404, Santa Cruz Biotechnology Inc., USA), p‐Chk2 (Thr68) (PA5‐104715, Invitrogen Inc. CA, USA), SOD‐2 (NB100‐1992, Novus Biologicals, USA), HSC70 (10654‐1‐AP, Proteintech, USA), p‐ULK1 (Ser757) (#14202, Cell Signaling Technology, USA) and ULK1 (sc‐390904, Santa Cruz Biotechnology, USA). β‐actin (AP0060, Bioworld Technology Inc., MN, USA) was loading control for total protein.
4
Antibody Panel for Stress Response
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Antibodies for IRE1α (#3294), PDI (#3501), PERK (#3192), eIF2α (#5324) and its phosphorylated form at ser51 (#3398, p-eIF2αser51) GRP78 (used in the cancer experiment, #3177), P62 (#5114), Bcl-2 (#3498), p-ULK1ser757 (#14202), ULK1 (#8054), p-JNKThr183/Tyr185 (#4668), JNK (#9252), ATF4 (#11815), Beclin-1 (#3495), caspase 12 (#2202), and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology. GAPDH (ab9485) antibody was purchased from Abcam (Cambridge, United Kingdom). LC3I and LC3II were measured by antibody (L7543) that was purchased from Sigma-Aldrich (St. Louis, MO, United States). Monoclonal primary antibodies were used for the detection of heat shock protein 70 (HSP70, StressGen, SPA-810), heat shock protein 60 (HSP60, StressGen, SPA-806), heat shock protein 90 (HSP90, StressGen, SPA-835), heat shock protein 47 (HSP47, StressGen, SPA-470) and thioredoxin interacting protein (TXNIP and MBL). Polyclonal primary antibodies were used to detect thioredoxin (TRX, IMCO, and ATRX-06), actin (Sigma, A-2066), heat shock protein 25 (HSP25, StressGen, and SPA-801), glucose-regulated protein 78 (GRP78, StressGen, SPA-826, used in the acute experiments). Horseradish peroxidase conjugated IgG secondary antibodies were used (Jackson ImmunoResearch Laboratories, PA, United States and StressGen and Zymed, San Francisco, CA, United States).
5
Western Blot Analysis of Autophagy Markers
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Following treatment, cells were immediately placed on ice and washed with ice-cold PBS. All the wash buffers and the final cell lysis/re-suspension buffers included 1X cOmpleteTM Mini Protease Inhibitor Cocktail (Roche, 11836153001), NaF (5 mM) (Sigma Aldrich, 201154) and Na3VO4 (200 μM) (Sigma Aldrich, S6508). Total cell proteins were resolved by electrophoresis on 8–12% SDS-polyacrylamide gels and transferred by electroblotting to PVDF membranes (EMD Millipore, IPVH00010). The membranes were blocked with 5% non-fat dry milk in TBST (1% Tween 20 in TBS) and incubated overnight at 4 °C in 5% non-fat dry milk in TBST with the antibodies against LC3 (1:1000, 12741), BECN1 (1:1000, 3495), SQSTM1/p62 (1:1000, 5114), ATG12 (1:1000, 4180), pULK1 Ser757 (1:1000, 14202), or ULK1 (1:1000, 8054) (Cell Signaling Technology). The membranes were also incubated with primary antibodies against, TonEBP/NFAT5 (1:1000, Novus Biologicals, NB120–3446), pULK1 Ser777 (1:500, EMD Millipore, ABC213), or β-Tubulin (1:10,000, DSHB, E7). Immunolabeling was detected using the AmershamTM ECLTM Prime Western Blotting Detection Reagent (Thermo Fisher Scientific, 45–002–401). All Western blot experiments were performed three independent times.
6
Metformin and AMPK Regulation of Apoptosis
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After the treatment with metformin (2 mM), glucose (15 mM) and/or siAMPK (50 nM)/siControl (50 nM) or Compound C (1 μM) at 24 h or 48 h, the cells were lysed in ice-cold NP-40 buffer containing protease inhibitor (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). The whole-cell lysates were incubated with protein A/G Sepharose beads (Roche) for 2 h at 4 °C, and the beads were discarded to eliminate non-specific binding. Then, the supernatants were incubated overnight with a Bcl-xl antibody (Cell Signaling Technology, Danvers, USA) at 4 °C, followed by incubation with protein A/G Sepharose beads for another 2 h at 4 °C. Then, western blotting was performed with the indicated antibodies according to standard protocols as previously described20 (link). The primary antibodies, including p-AMPK (T172) (#50081), phospho-Acetyl-CoA carboxylase (p-ACC) (#11818), phospho-Tuberin/TSC2 (p-TSC2) (Ser1387) (#23402), phospho-Raptor (p-Raptor) (Ser792) (#2083), p-mTOR (S2448) (#5536), phospho-p70 S6 kinase (p-S6K1) (Thr389) (#97596), p-ULK1 (Ser757) (#14202), p-4E-BP1 (Thr70) (#9455), cleaved Caspase-3 (Cl-Caspase3) (Asp175) (#9664), p-Akt (Ser473) (#4060), p-Akt (Thr308) (#13038), RagB (#8150), LC3B (#3868), β-Actin (#3700) were all purchased from Cell Signaling Technology, Danvers, USA.
7
Western Blot Analysis of Autophagy Markers
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Proteins were obtained from the hearts or cell extracts and were analysed using Western blot analysis, as previously described [33 (link)]. The following primary antibodies were used: anti-LC3 (1:500; Abcam, Cambridge, MA, UK), anti-p62 (1:500; Abcam), anti-Beclin1 (1:500; Abcam), anti-AMPK (1:1000; Cell Signaling, Danvers, MA, USA), anti-p-AMPK-Thr172 (1:1000; Cell Signaling), anti-mTOR (1:1000; Cell Signaling), anti-p-mTOR-Ser2448 (1:1000; Cell Signaling), ULK1 (1:1000; Cell Signaling), and p-ULK1-Ser757 (1:1000; Cell Signaling). Targeted bands were normalized to GAPDH to confirm equal protein loading. The Western blot bands were quantified using ImageJ 3.0 software.
8
Western Blot Analysis of Signaling Proteins
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Total infected B-cell protein was obtained through lysis with RIPA buffer. Samples in acrylamide gel were resolved at 12% or 15% via SDS-PAGE. Subsequently, proteins were transferred to a PVDF membrane and then blocked using 1% BSA solution for 1 h. Antibodies were diluted in block solution: pS6 ser 235/236 (Cell, Danvers, MA, USA D57.2.2E), 1:1000; S6 (Cell signaling, 5G10), 1:1000; pULK1 ser 757 (Cell signaling, D7O6U), 1:1000; ULK (Cell signaling, D8H5), 1:1000; LC3 (Cell signaling, #2775), 1:1000; β actin (Cell signaling, D6A8), 1:2000. The membranes, together with the primary antibodies, were incubated for 1 h at room temperature. After incubation, the membranes were washed five times with TBST. Then, antirabbit HRP at 1:3000 dilution (Cell signaling, #7074) was added. Five washes with TBST were performed to eliminate antibody excess. Images were obtained in ChemiDoc (BioRad, Hercules, CA, USA) and analyzed by Image Lab (BioRad, Hercules, CA, USA, version 6.1).
9
Western Blot Analysis of Autophagy Proteins
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At times of post-infection, monolayers were washed with ice-cold PBS and then scraped in NP40 lysis buffer (25 mmol/L Tris HCl, 150 mmol/L NaCl; 1 mmol/L EDTA; 5 mmol/L EGTA; 1 mmol/L MgCl2; 10% Glycérol; 1% NP40; Plus complete protease inhibitor cocktail, Roche; 5 µL/mL PMSF; 10 µL/mL Sodium Orthovanadate; 5 mmol/L NEM, Sigma Aldrich, Saint-Louis, MO, USA). Whole cell extracts were subjected to SDS-PAGE on 12% or 18% acrylamide gels. Proteins were electroblotted onto nitrocellulose membranes (Amersham International, Little Chalfont, UK) and the membranes were immunoblotted for IRGM (rabbit, 1:1000, Abcam, Cambridge, UK), SQSTM1/p62 (mouse, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), ULK-1, p-ULK-1 Ser757, LC3A/B and GAPDH as internal control (all rabbit, 1:1000, Cell signaling technology, Danvers, MA, USA). Immuno-reactants were detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G antibody, ECL reagents (Abcam, Cambridge, UK), through a ChemiDoc camera (Bio Rad, Hercules, CA, USA). Image J software was used to estimate protein quantity. Results were expressed as protein amount relative to GAPDH and to uninfected or transfected against siCTL MDM.
10
Autophagy-related Protein Expression Analysis
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Antibodies against the following proteins were used: Sequestosome 1 (p62; 1:1,000; cat. no. ab109012; Abcam), Beclin1 (1:1,000; cat. no. ab210498; Abcam), cleaved caspase-3 (1:1,000; cat. no. AB3623; Sigma-Aldrich; Merck KGaA), cleaved caspase-9 (1:1,000; cat. no. AB3629; Sigma-Aldrich; Merck KGaA), microtubule-associated protein light chain 3 A/B (LC3A/B; 1:1,000; cat. no. 4108; Cell Signaling Technology, Inc.), AMPK (1:1,000; cat. no. 5831; Cell Signaling Technology, Inc.), phosphorylated (p)-AMPK (Thr172) (1:1,000; cat. no. 2535; Cell Signaling Technology, Inc.), Unc-51 like autophagy activating kinase 1 (ULK1; 1:1,000; cat. no. 6439; Cell Signaling Technology, Inc.), p-ULK1 (Ser757) (1:1,000; cat. no. 14202; Cell Signaling Technology, Inc.) and β-actin (1:5000; cat. no. sc-47778; Santa Cruz, Biotechnology. Inc.). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse/rabbit IgG) (1:5,000; cat. nos. 7076 and 7074; Cell Signaling Technology, Inc.) were used.
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