Islet proteins (∼150) lysed in RIPA buffer with protease inhibitors (Millipore) were separated by SDS-PAGE and blotted with anti-PGC-1α (1:1000, Calbiochem 4Cl.3), anti-ATGL (1:1000, Cell Signaling #2138) and anti-HSL (1:1000, Cell Signaling #4107).
Anti hsl
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-HSL is a primary antibody produced by Cell Signaling Technology that specifically recognizes Hormone-Sensitive Lipase (HSL), an enzyme involved in the hydrolysis of stored triglycerides. This antibody can be used for the detection and analysis of HSL in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti atgl, by Cell Signaling Technology (8 mentions)
Anti β actin, by Merck Group (4 mentions)
Insulin, by Merck Group (3 mentions)
Anti pparγ, by Santa Cruz Biotechnology (3 mentions)
Anti p hsl, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Anti ire1α, by Cell Signaling Technology (2 mentions)
Anti p hslser660, by Cell Signaling Technology (2 mentions)
Anti eif2α, by Cell Signaling Technology (2 mentions)
Anti c ebpα, by Cell Signaling Technology (2 mentions)
Anti adiponectin, by Cell Signaling Technology (2 mentions)
Anti phospho hsl ser660, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Anti cpt1a, by Abcam (1 mentions)
Total oxphos complex kit, by Thermo Fisher Scientific (1 mentions)
Anti α sma, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ecl plus western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
21 protocols using anti hsl
1
Islet Protein Expression Analysis
2
Immunoblotting of Cell Lysates
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Cell lysates were prepared as previously described [28 (link)]. Cell lysates were subjected to SDS-PAGE, transferred to PVDF membranes (Merck Millipore), and then immunoblotted with antibodies for anti-ATGL (#2138), anti-HSL (#4107), and anti-GAPDH (#2118) obtained from Cell Signaling Technology (Danvers, MA), Total OxPhos Complex Kit (# 458099) from Invitrogen (Life Technologies Australia Pty Ltd), anti-14-3-3 (sc-33752) from Santa Cruz Biotech (Dallas, TX), and anti-CPT1A (#ab128568) from Abcam (Cambridge, MA).
3
Immunoblotting Analysis of Protein Expression
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Analysis of protein expression by immunoblotting was performed as previously described (10 (link)). Proteins of cell lysates (20–40 μg) were dissolved in SDS sample buffer, separated by 10% or 12.5% SDS-PAGE, and transferred onto a polyvinylidene fluoride membrane (Carl Roth GmbH, Karlsruhe, Germany). The membrane was blocked with 10% nonfat dry milk and incubated with the following primary antibodies: anti-ATGL, anti-HSL, anti-GAPDH, and anti-calnexin from Cell Signaling Technology (Danvers, MA; 2138S/ATGL, 4107S/HSL, 2118S/GAPDH, 2679S/calnexin), anti-6x HIS tag, and anti-NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) from Abcam (Cambridge, England; ab18184/6x HIS-tag®, ab157221/NDUFS1), anti-α-SMA from Thermo Fisher Scientific (Waltham, MA; PA5-22251/α-SMA), and anti-KIAA1363 from Invitrogen GmbH (PA5-50285/NCEH1 = KIAA1363), respectively. For detection, membranes were incubated with horseradish peroxidase-labeled secondary antibodies specific for respective primary antibody. Bands were visualized using the ECL plus Western blotting Detection Reagent (Thermo Fisher Scientific) and ChemiDoc Touch Imaging System (Bio-Rad).
4
Characterization of LDAH Antibodies
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Custom polyclonal rabbit anti-human and mouse LDAH antibodies were generated at Bethyl Laboratories, and characterized as previously described15 (link). Bodipy 493/503, LipidTox, and RNAiMAX were from Invitrogen. Fugene HD was purchased from Promega. Isoproterenol hydrochloride, anti-beta actin antibody, anti Flag-M2-HRP, anti Flag-M2-agarose, mouse IgG, oleic acid, 3-isobutyl-1-methylxanthine, insulin, dexamethasone and nocodazole were from Sigma-Aldrich. Triacsin C and anti-ubiquitin antibody were from Santa Cruz Biotechnology. Anti-PLIN1 antibody was from Progen. Anti-beta tubulin and anti-PLIN2 antibodies were from Nobus Biologicals. Anti-PLIN3 was purchased from Fitzgerald. Anti-ATGL and anti-HSL were from Cell Signaling. Alexa Fluor 647-conjugated secondary antibodies and anti-GM130 were from BD Pharmingen. Protein G-agarose and protein-A-agarose were from Thermo Scientific. MG132 was purchased from Calbiochem. Total and free cholesterol were measured using kits from Wako. TAG were determined with Infinity Trglyceride reagents from Thermo Scientific. 14C-oleic acid was purchased from Perkin Elmer.
5
Adipose Tissue Protein Analysis
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The following antibodies were purchased from Cell Signaling Technology: anti-ATGL (#2439, 1:1000, RRID:AB_2167953), anti-Phospho-HSL (#4137,1:1000, RRID:AB_2135498), anti-HSL (#4107,1:1000, RRID:AB_2296900), and anti-Phospho-PKA (#9621,1:1000, RRID:AB_330304). The following antibodies were purchased from Abcam: anti-cAMP (ab76238,1:1000, RRID:AB_1523259) and anti-UCP1 (ab234430,1:1000, RRID:AB_2905638).
6
Western Blot Analysis of Cell Signaling
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Western blot was performed as previously described [21 (link)]. Primary antibodies, anti-IRE1α, anti-HSL, anti-p-HSLSer660, anti-eIF2α, anti-p-eIF2αSer51, anti-BiP and anti-p-PKA substrates (all from Cell Signaling Technology, MA, USA), anti-UCP1 and anti-ATF4 (Santa Cruz Biotechnology) and anti-β-actin (Sigma, MO, USA) were incubated at 4°C overnight, and specific proteins were visualized by ECL Plus (GE Healthcare, Buckinghamshire, UK).
7
Western Blot Analysis of Adipose Tissue Proteins
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Expression of HSL, its serine-563-phosphorylated form (HSL Ser563) and ATGL were measured in epididymal fat pad homogenates by western blot. Antibodies for immunoblotting were as follows: anti-HSL (1:2000, Cell Signaling Technology, USA), anti-HSL Ser563 (1:500, Cell Signaling Technology), anti-ATGL (1:2000, Cell Signaling Technology) and anti-β-actin (1:5000, Sigma). Epididymal adipose tissue homogenates (25 µg total protein per well) were separated by SDS page, and proteins transferred to a polyvinylidene fluoride membrane. Membranes were then blocked [5% milk and 0.1% TWEEN-20 in PBS (PBS-T)], and incubated at 4°C overnight in primary antibodies diluted in 1% BSA in PBS-T. After being incubated with HRP-conjugated secondary antibodies (1:500, Wako Chemicals) in 5% milk in PBS-T, blots were developed using an Enhanced Chemiluminescent Detection Kit (GE Healthcare). Digital images of protein bands were acquired (ImageQuant 350, GE Healthcare, UK) and semi-quantitative analysis of protein content performed by densitometry using ImageQuant TL software (GE Healthcare, UK), with values normalised to β-actin as a loading control.
8
Western Blot Analysis of Phosphorylated HSL
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Whole-tissue protein lysate was extracted in RIPA buffer (G-Biosciences) containing Halt proteinase inhibitor (Thermo Fisher Scientific, 78430) and phosphatase inhibitor (Thermo Fisher Scientific, 78420). Protein lysate was determined using the bicinchoninic acid assay (Pierce). Protein lysates were denatured in Laemmli buffer (BioRad), resolved by 4–12% Mini-PROTEAN TGX SDS–PAGE (BioRad) and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies diluted in EveryBlot blocking buffer (BioRad) overnight at 4 °C and then incubated with secondary antibody anti-rabbit HRP (Jackson Immuno Research, 711-036-152, 1:10,000) or anti-mouse HRP (Jackson Immuno Research, 715-036-150, 1:10,000) diluted in EveryBlot at room temperature. The results were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (Invitrogen). The following antibodies were used for immunoblotting: anti-p-HSL(Ser660) (Cell Signaling, 45804, 1:1,000), anti-HSL (Cell Signaling, 4107, 1:1,000), anti-α-tubulin (Abcam, 7291, 1:10,000). Specifically, HSL was blotted after stripping the p-HSL membrane.
9
Adipogenesis Regulation by Bilobalide
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Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco (Rockville, MD, USA). Penicillin-streptomycin solution was purchased from Hyclone (Provo, UT, USA). Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3T3-L1 preadipocytes were purchased from ATCC (Manassas, VA, USA). Bilobalide was obtained from the National Institutes for Food and Drug Control (Beijing, China). Primary antibodies specific for anti-pACC1 (S79), anti-ACC1, anti-FASN, anti-perilipin A, anti-ATGL, anti-C/EBPα, anti-PPARγ, anti-HSL, anti-pHSL (S563), anti-GLUT-4, anti-CPT-1α, anti-AMPK, and anti-pAMPK (T172) were acquired from Cell Signaling Technology (Danvers, MD, USA). Anti-SREBP-1c was acquired from Abcam (Cambridge, UK). Anti-β-Actin and goat anti-mouse and goat anti-rabbit IgG secondary antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). The AMPK assay kit was purchased from Cell Signaling Technology.
10
Protein Extraction and Western Blot Analysis in Adipocytes
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Protein extraction and Western blot analysis were performed as described previously33 (link) with some modifications. Adipocytes and adipose tissues were lysed by dissolution in lysis buffer. The proteins obtained were subjected to sodium dodecyl sulfate–polyacrylamides gel electrophoresis, following which the separated protein bands were transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with the following primary antibodies: anti-PRMT4 (1:2000, Cell Signaling Technology, USA), anti-phosphorylated HSL (1:2000, Cell Signaling Technology, USA), anti-HSL (1:1000, Cell Signaling Technology, USA), and anti-beta (β)-tubulin (1:1000, Cell Signaling Technology, USA). Then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies. The specific bands were detected using enhanced chemiluminescence detection reagents with a Bio-Rad (Hercules, CA, USA) imaging system.
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