Phospho hsl ser563

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-HSL (Ser563) is a primary antibody developed by Cell Signaling Technology. It is designed to detect the phosphorylation of hormone-sensitive lipase (HSL) at serine 563. This modification of HSL plays a role in the regulation of lipid metabolism.

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Lab products found in correlation

Phospho hsl ser660, by Cell Signaling Technology (4 mentions) Phospho acc ser79, by Cell Signaling Technology (2 mentions) Phospho akt ser473, by Cell Signaling Technology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Colorimetric assay, by Fujifilm (1 mentions) Colorimetric assay, by Merck Group (1 mentions) Gapdh and β actin, by Abcam (1 mentions) Insulin receptor β, by Santa Cruz Biotechnology (1 mentions) Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions) Phospho creb ser133, by Cell Signaling Technology (1 mentions) Tfiib, by Santa Cruz Biotechnology (1 mentions) Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (1 mentions) Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions) Amido black staining, by Merck Group (1 mentions) Phospho ser thr pka substrate, by Cell Signaling Technology (1 mentions) Fabp4, by Cayman Chemical (1 mentions) Ez ecl enhanced chemiluminescence detection kit for hrp, by Sartorius (1 mentions) Phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions) Cl xposure film, by Thermo Fisher Scientific (1 mentions) Pierce ecl substrate, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Phospho-HSL (9 citations) Anti-phospho-Ser563 HSL (2 citations)

12 protocols using phospho hsl ser563

Plasma Lipid and Insulin Signaling Analysis

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Plasma TGs and free glycerol levels were measured with a colorimetric assay (Sigma-Aldrich). Plasma free FA levels were measured with a colorimetric assay (Wako Chemicals, Neuss, Germany). Plasma insulin was measured by an ELISA kit (Mercodia, Uppsala, Sweden). Blood glucose levels were measured with a strip-based glucose meter.
Western blots were performed as previously described (28 (link)). Primary antibodies against Acc, phospho-Acc Ser⁷⁹, Atpcl, phospho-Atpcl Ser⁴⁵⁵, phospho-Akt Ser⁴⁷³, phospho-Hsl Ser⁵⁶³, and phospho–insulin receptor β (all Cell Signaling Technology); GAPDH and β-actin (both Abcam); insulin receptor β (Santa Cruz Biotechnology); and FA synthase (BD Bioscience) were used.

Scherer T., Lindtner C., O’Hare J., Hackl M., Zielinski E., Freudenthaler A., Baumgartner-Parzer S., Tödter K., Heeren J., Krššák M., Scheja L., Fürnsinn C, & Buettner C. (2016). Insulin Regulates Hepatic Triglyceride Secretion and Lipid Content via Signaling in the Brain. Diabetes, 65(6), 1511-1520.

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Immunoblotting Analysis of Cellular Signaling

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Preparation of whole-cell extracts and immunoblotting were done essentially as described [32 (link)]. Briefly, protein lysates were separated on NuPAGE 4–12% Bis-Tris gradient gels (Life Technologies) using NuPAGE MOPS SDS Running Buffer (Life Technologies) and transferred by semi-dry blotting onto polyvinylidene diflouride membrane (GE Healthcare). Equal loading and transfer were confirmed by Amido Black staining (Sigma Aldrich). All washing and incubation steps were carried out with Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat dry milk or BSA. Primary antibodies used were: AKT (#9272), phospho-CREB (Ser133) (#9198), phospho-GSK3α (Ser21) (#9316), phospho-GSK3β (Ser9) (#5558), phospho-p38 MAPK (Thr180/Tyr182) (#9211), phospho-MKK3/6 (Ser189/Ser207) (#12280), phospho-HSL (Ser660) (#4126), phospho-HSL (Ser563) (#4139), phospho-(Ser/Thr) PKA substrate (#9621) (all from Cell Signaling Technology), TFIIB (#sc-225) (Santa Cruz Biotechnology), CYC1 (#sc-7159) (Santa Cruz Biotechnology), FABP4 (#10004944) (Cayman Chemical) and UCP1 (#10983) (Abcam). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit or anti-mouse (DAKO). EZ-ECL Enhanced Chemiluminescence Detection Kit for HRP (Biological Industries) was used for detection.

Markussen L.K., Isidor M.S., Breining P., Andersen E.S., Rasmussen N.E., Petersen L.I., Pedersen S.B., Richelsen B, & Hansen J.B. (2017). Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor. PLoS ONE, 12(9), e0185624.

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Adipose Tissue Protein Extraction and Immunoblotting

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Adipose tissue (200-300 mg) was homogenized in 1 ml of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with protease inhibitors and phosphatase inhibitors (Roche). The homogenate was centrifuged at 10,000 ×g for 20 min at 4°C. The clear phase was collected and centrifuged again at 13,000 ×g for 10 min. Protein concentrations were measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein samples were separated by 8% SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes. The membranes were immunoblotted first with 1:1000 diluted antibody for phosphorylated hormone sensitive lipase (Phospho-HSL, Ser563) (Cell Signaling, Danvers, MA, USA) and then with 1:1000 diluted antibody for total HSL (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies were detected by 1:1000 diluted horseradish peroxidase-coupled IgG (Santa Cruz Biotechnology) followed by Pierce ECL substrate (Thermo Scientific). Chemiluminescent signals were detected by exposing the membrane to a CL-Xposure film (Thermo Scientific, Rockford, IL, USA).

Zhao L, & Jiang H. (2023). Growth hormone stimulates lipolysis in mice but not in adipose tissue or adipocyte culture. Frontiers in Endocrinology, 13, 1028191.

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Comprehensive Protein Immunoblot Analysis

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Tissues were homogenized and lysed in lysis buffer as previously described [40 (link)]. The protein concentration was determined by Bradford protein assay (Bio-Rad). Equal amounts of proteins were loaded and immunoblot analyses were carried out according to standard protocol. The following antibodies from Cell Signaling Technologies were used: HSL (4107), Phospho-HSL (Ser563) (4139), ATGL (2138), HSP60 (12165), DRP1 (5391), phosphor-DRP1 Ser616 (4494), phosphor-DRP1 Ser637 (4867), phosphor-AKT Ser473 (4060), AKT (9272), and LC3A/B (4108). Total OXPHOS Rodent WB Antibody Cocktail (ab110413), MLKL (ab172868), Anti-RIP3 (phosphor T231 + S232) (ab201912), and UCP1 (ab1426) were obtained from Abcam. GAPDH (60004-1-IG) and P62 (18420-1-AP) were obtained from Proteintech; PLIN1 (GP29, Progen Biotechnik GmbH); β-actin (MAB1501, MilliporeSigma); RIPK3 (NBP1-77299SS, Novus Biologicals).

Zhou H., Xu C., Lee H., Yoon Y, & Chen W. (2020). Berardinelli–Seip congenital lipodystrophy 2/SEIPIN determines brown adipose tissue maintenance and thermogenic programing. Molecular Metabolism, 36, 100971.

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Western Blot Analysis of Epididymal WAT Proteins

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Proteins from epididymal WAT were extracted and quantified and western blots carried out as in Ref. [26] (link). Membranes were incubated with the following primary antibodies: β3-adrenergic receptor (1:2000, catalog # 15688, Millipore, Molsheim, France), adipose triglyceride lipase (ATGL, 1:1000, catalog # 2439, Cell Signaling), hormone-sensitive lipase (HSL, 1:1000, catalog # 4107, Cell Signaling), phospho-HSL ser 563 (1:1000, catalog # 4139, Cell Signaling), phospho-HSL ser 660 (1:1000, catalog # 4126, Cell Signaling) and β-actin (1:4000, catalog # 4967, Cell Signaling). β-actin was used as loading control. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL plus, PerkinElmer) then exposed on radiographic films. Bands quantification was performed using ImageJ software (NIH, Bethesda, MA).

Cardinal P., André C., Quarta C., Bellocchio L., Clark S., Elie M., Leste-Lasserre T., Maitre M., Gonzales D., Cannich A., Pagotto U., Marsicano G, & Cota D. (2014). CB1 cannabinoid receptor in SF1-expressing neurons of the ventromedial hypothalamus determines metabolic responses to diet and leptin. Molecular Metabolism, 3(7), 705-716.

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Western Blot Analysis of Cell Signaling

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Tissue or cells were lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails. Western blots were probed with the following antibodies: total JAK2, total STAT5, phospho-JAK2 (Tyr1007/1008) phospho-STAT5 (Tyr694) , ATGL , HSL and phospho-HSL (Ser563) purchased from Cell Signaling Technology (Danvers, MA, USA); EPOR (R&D Systems, Minneapolis, MN, USA); HIF-1α (Sigma-Aldrich, Saint Louis, MO, USA); HIF-2α (R&D Systems) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Li J., Yang M., Yu Z., Tian J., Du S, & Ding H. (2019). Kidney-secreted erythropoietin lowers lipidemia via activating JAK2-STAT5 signaling in adipose tissue. EBioMedicine, 50, 317-328.

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Adipose Tissue Protein Expression

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The freshly extracted adipose tissues were quickly frozen in liquid nitrogen. The frozen samples were kept at −80 °C freezer or on ice for immediate homogenization. Briefly, 800 μl ice-cold lysis buffer was added in the tube per 100 mg of tissue from each animal, and vortexed on ice for 2 h. Then the tubes were centrifuged at 14,000 × rpm at 4 °C for 30 min in a microcentrifuge. The supernatants were aspirated and transferred to a fresh tube on ice. A small volume of lysate was used for protein quantification assay to determine the protein concentration for each lysate. The primary antibodies against phospho-HSL (Ser563) (Cell Signaling Technology; #4139; Lot#4), p-HSL (Ser660) (PA5–64494; Lot#3010125; Invitrogen) and HSL (Cell Signaling Technology; #4107; Lot#3) were diluted at 1:1000. The protein expressions were detected using traditional western blots or automated complete protein analysis solutions (Jess; protein simple). The secondary antibodies used with Jess included anti-rabbit secondary NIR antibody (043–819; protein simple) and anti-mouse secondary IR antibody (043–822; protein simple).

Zhang J., Hou Y., Du X.L., Chen D., Sui G., Qi Y., Licinio J., Wong M.L, & Yang Y. (2020). ADORA1-driven brain-sympathetic neuro-adipose connections control body weight and adipose lipid metabolism. Molecular psychiatry, 26(7), 2805-2819.

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Western Blot Analysis of Lipid Metabolism Proteins

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Liver nuclear extracts were prepared as described previously23 (link). Western blotting was performed using specific antibodies against ATGL (Cell Signaling, #2138), mouse CREBH66 (link), HMGCS (Santa Cruz, sc-33828), HSP90 (Santa Cruz, sc-7947), Lamin B1 (Santa Cruz, sc-56145), HSL (Cell Signaling, #4170), phospho-HSL (Ser563) (Cell Signaling, #4139), phospho-HSL (Ser660) (Cell Signaling, #4126), perilipin (Cell Signaling, #9346), and PPARα (Santa Cruz, sc-9000). Following incubation with secondary antibodies, protein bands were visualized by SuperSignal West Pico chemiluminescence substrate (Thermofisher), imaged using Flurochem E system (Proteinsimple, CA, USA), and quantitated using Alphaview software (Proteinsimple, CA, USA).

Park J.G., Xu X., Cho S., Hur K.Y., Lee M.S., Kersten S, & Lee A.H. (2016). CREBH-FGF21 axis improves hepatic steatosis by suppressing adipose tissue lipolysis. Scientific Reports, 6, 27938.

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Comprehensive Protein Analysis via Western Blot

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Whole cell lysates were prepared with CHAPS buffer [40 mM HEPES (pH7.5), 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS, 50 mM NaF, 1.5 mM Na₃VO₄, 10 mM glycerophosphate, 10 mM pyrophosphate, 1 mM PMSF] supplemented with protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche Diagnostics). Protein concentration was measured using the BCA assay (Thermo Scientific Pierce). Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore) using the wet transfer system (Bio-Rad). Membranes were treated with BlockingOne blocking buffer (Nacalai Tesque) for 1 hr and incubated with primary antibody in Can Get Signal (Toyobo) overnight. After 3 washes in TBS-T (Tris buffered saline (pH 7.4) with 0.1% Tween 20), membranes were incubated with secondary antibodies against mouse or rabbit IgG with horseradish peroxidase in Can Get Signal for 1 hr. ECL Prime Western Blotting Detection reagent (Amersham) was used to detect immune complexes. Images were analyzed by Amersham Imager 600 (GE Healthcare Life Science). Primary antibodies recognizing the following proteins were used: Utx, phospho-HSL (Ser563), HSL, ATGL (all from Cell Signaling Technologies); beta-actin (Santa Cruz).

Ota K., Komuro A., Amano H., Kanai A., Ge K., Ueda T, & Okada H. (2019). High Fat Diet Triggers a Reduction in Body Fat Mass in Female Mice Deficient for Utx demethylase. Scientific Reports, 9, 10036.

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Ceramide Modulation of β-Adrenergic Signaling

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Differentiated adipocytes were treated with 50 μM of C₂-ceramide or C₆-dihydroceramide for 4 h. The cells were then treated with 10 μM of isoproterenol for 30 min, and cells were analyzed for β-adrenergic signaling intermediates pHSL, HSL, and pPKA substrates by western blot. To inhibit PP2A activity, following 4 h of treatment with 50 μM of C₂-ceramide, the cells were treated with 25 nM of microcystin-LR for 1 h. The cells were then treated with isoproterenol as mentioned above. HSL phosphorylation was assessed via immunoblotting. The following antibodies were used: phospho-HSL (Ser563), Cell Signaling, catalog number: 4139, HSL, Cell Signaling, catalog number: 4107, phospho-PKA: phospho-PKA Substrate (RRXS∗/T∗) (100G7E), Cell Signaling, catalog number: 9624, β-actin: beta-actin (8H10D10), Cell Signaling, catalog number: 3700S.

Chaurasia B., Ying L., Talbot C.L., Maschek J.A., Cox J., Schuchman E.H., Hirabayashi Y., Holland W.L, & Summers S.A. (2020). Ceramides are necessary and sufficient for diet-induced impairment of thermogenic adipocytes. Molecular Metabolism, 45, 101145.

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