Samples of frozen SC-WAT were homogenized in lysis buffer with 1 M HEPES, 2 M NaCl, 20% SDS, 0.5 M EDTA, 100mM Benzamidina (pH = 7.4), protease and phosphatase inhibitor cocktail EDTA-free (Thermo Scientific) at the concentration of 10 μl/ml. Samples were centrifuged for 15 min at 15.000 rpm at 4°C. Protein concentrations of the homogenates were measured by the BCA method with a protein assay kit (PIERCE Biotechnology, Rockford, IL, USA) using bovine serum albumin as a standard. Aliquots (60 μg) of protein were subjected to SDS-PAGE. The membranes were incubated overnight at 4°C with the following primary antibodies: p- AKT (1:1000), t-AKT (1:1000), GLUT-4 (1:1000), AT2 (1:1000), MAS (1.25:1000) (Abcam, Cambridge, USA), p-HSL (1:1000), t-HSL (1:1000), Perilipin (1:1000), ATGL (1:1000), AT1 (1:1000), (Cell Signaling, Beverly, MA) and beta-actin (Abcam, Cambridge, USA). The signal on the membrane was detected via the peroxidase reaction in the ECL solution using an Image Quant LAS 4000 mini system (GE Healthcare Life Sciences). Band intensities were quantified based on optical densitometry measurements using the Image J program (version 1.43 for Windows).
T hsl
Manufactured by Cell Signaling Technology
Sourced in United States
T-HSL is a laboratory equipment product offered by Cell Signaling Technology. It functions as a tool for the detection and analysis of hormone-sensitive lipase (HSL) in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000 mini system, by GE Healthcare (1 mentions)
Protein assay kit, by Thermo Fisher Scientific (1 mentions)
Glut4, by Abcam (1 mentions)
P akt, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Perilipin, by Cell Signaling Technology (1 mentions)
P hsl, by Cell Signaling Technology (1 mentions)
T akt, by Abcam (1 mentions)
Protease and phosphatase inhibitor cocktail edta free, by Thermo Fisher Scientific (1 mentions)
Las 4000, by Cytiva (1 mentions)
Protease phosphatase inhibitor cocktail, by Roche (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
P hsl ser563, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Flag tag (flag), by GenScript (1 mentions)
T akt, by Cell Signaling Technology (1 mentions)
2 protocols using t hsl
1
Protein Extraction and Analysis from Frozen Subcutaneous White Adipose Tissue
2
Western Blot Analysis of Cellular Proteins
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Tissue was homogenized, and protein was extracted using radioimmunoprecipitation lysis buffer (Beyotime) containing phosphatase/protease inhibitor cocktail (Roche). Protein concentration was measured using the BCA protein assay kit (Beyotime). Equivalent amount of protein was loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred onto a nitrocellulose membrane (MilliporeSigma). The membrane was blocked with 5% skim milk for 1 h at room temperature, and incubated overnight at 4 °C with primary antibody against CLSTN3 (Proteintech, 13302-1-AP), CAV1 (Proteintech, 66067-1-Ig), Actin (Abcam, ab8227), Tubulin (MilliporeSigma, T6199), UCP1 (Abcam, ab10983), P-AKTThr308 (Cell Signaling Technology, 13,038), T-AKT, (Cell Signaling Technology, 9272), P-HSLSer563 (Cell Signaling Technology, 4139), T-HSL (Cell Signaling Technology, 4107), FLAG (GenScript, AF519), HA (Cell Signaling Technology, 3724), APP (GeneTex, GTX101336), VDAC1 (Abcam, ab154856), and OXPHOS rodent WB cocktail (Abcam, ab110413). Then, the membrane was incubated with secondary anti-rabbit or anti-mouse antibody for 1 h at room temperature. Protein bands were detected with ECL chemiluminescent reagent (MilliporeSigma) using the Image Quant LAS4000 System (GE Healthcare).
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