Western blotting was performed, as previously described [20 (link)]. Western blot analysis was performed using primary antibodies against phosphorylated hormone-sensitive lipase (p-HSL Ser660, rabbit, Cell Signaling), hormone-sensitive lipase (HSL, rabbit, Cell Signaling), phosphorylated perilipin1 (p-PLIN1 Ser522, mouse, VALA science), perilipin1 (PLIN1, rabbit, Santacruz), beta-actin (mouse, Santacruz), phosphor-(Ser/Thr) protein kinase A substrates (p-PKA substrates, rabbit, Cell Signaling), sirtuin 1 (SIRT1, rabbit, Cell Signaling), Estrogen Receptor Alpha Antibody A300-498A (Estrogen receptor alpha (ERα), rabbit, Bethyl laboratories INC.), adipose triglyceride lipase (ATGL, rabbit, Cell Signaling), phosphor- cAMP-response element binding protein (p-CREB ser133, rabbit, Cell Signaling), cAMP-response element binding protein (CREB, rabbit, Cell Signaling), uncoupling protein 1 (UCP1, rabbit, Alpha Diagnostic International), total oxphos rodent WB antibody cocktail (mouse, Abcam: ATP synthase subunit alpha, mitochondrial (ATP5A), succinate dehydrogenase [ubiquinone] iron–sulfur subunit, mitochondrial (SDHB), NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial (NDUFB8), cytochrome b-c1 complex subunit 2, mitochondrial (UQCRC2)), and α/β tubulin (rabbit, Cell Signaling). Blots were visualized with Super Signal West Dura Substrate (Pierce, Invitrogen).
Phsl ser660
Manufactured by Cell Signaling Technology
Sourced in United States
The PHSL Ser660 is a laboratory product designed to detect and quantify the phosphorylation of the serine residue at position 660 in the PHSL protein. It is a tool used in cellular and molecular biology research to study signaling pathways and protein modifications.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 antibody, by Merck Group (2 mentions)
Chemidoc mp imaging station, by Bio-Rad (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Western lightning, by PerkinElmer (2 mentions)
Phsl ser563, by Santa Cruz Biotechnology (2 mentions)
P perilipin, by Merck Group (2 mentions)
Fluorophore conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Erk1 2, by Santa Cruz Biotechnology (2 mentions)
P erk1 2, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
P creb ser133, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Ppka substrates, by Cell Signaling Technology (1 mentions)
Plin1, by Cell Signaling Technology (1 mentions)
Total oxphos rodent wb antibody cocktail, by Abcam (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
P p38 thr180 tyr182, by Cell Signaling Technology (1 mentions)
β actin, by Bioworld Technology (1 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
P p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
7 protocols using phsl ser660
1
Comprehensive Western Blot Analysis
2
Adipose Tissue Protein Analysis
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Epididymal adipose tissue was homogenized with a tissue grinder at 4°C temperature and the lysate were centrifugation at 12000 rpm for 20 minutes. The protein concentrations were measured with BCA kit (KeyGEN BioTECH Corp., Ltd, Nanjin, China). The protein samples were denatured, separated by SDS-PAGE and then transferred to the PVDF membrane. Membranes were blocked with 5% skim milk or bovine serum albumin (BSA) for 3 hours, and then incubated overnight at 4°C with specific primary antibodies as follows: ATGL (2138S, Cell Signaling Technology), P-HSL (ser660) (4126S, Cell Signaling Technology), HSL (4107S, Cell Signaling Technology), P-GSK-3β (ser9) (5558S, Cell Signaling Technology), GSK-3β (9315S, Cell Signaling Technology), β-actin (AP0060, Bioworld), PKA (5842S, Cell Signaling Technology), P-p38 (Thr180/tyr182) (9219S, Cell Signaling Technology), p38 (ab170099, Abcam), P44/42 MAPK (ERK1/2) (9102S, Cell Signaling Technology), P-P44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling Technology) and AT2R (ab92445, Abcam). Membranes were washed and incubated with secondary antibodies (BA1054, BOSTER Biological Technology) at room temperature for 3 hours. After washing, membranes were exposed by Super ECL Prime (SEVEN BIOTECH) with ChemiDoc™ Imaging System (BIO-RAD, USA). The images were analyzed quantitatively by densitometry with Image J software.
3
Palmitic Acid Metabolism Regulation
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Type II collagenase, isoproterenol, fatty acid (FA)-free bovine serum albumin (BSA), palmitic acid, and the free glycerol determination kit were purchased from Sigma (St. Louis, MO, USA). [1-14C] palmitic acid and D-[U-14C] glucose were purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Protease (cOmplete Ultra Tablets) and phosphatase (PhosSTOP) inhibitors were obtained from Roche Diagnostics GmbH (Mannheim, Germany). The β-actin (Cat # 4067), AMPK (Cat # 2532), pAMPKThr172 (Cat # 2535), AKT (Cat # 9272), pAKTSer473 (Cat # 9271), p38 (Cat # 9212), pp38 (Cat # 9211), hormone-sensitive lipase (HSL; Cat # 4107), and pHSLSer660 (Cat # 4126) antibodies were purchased from Cell Signalling (Danvers, MA, USA). The ACC (Cat # ab45174) and pACCSer79 (Cat # ab68191) antibodies were purchased from Abcam (Toronto, ON, Canada). ALY688 was provided by Allysta Pharmaceuticals, Belmont, CA, USA.
4
Western Blot Analysis of Signaling Proteins
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Protein samples were denatured in Laemmli buffer, and loaded onto SDS-PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MD). Membranes were then incubated overnight at 4 °C in wash buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20) containing 1% BSA with the primary antibody. Antibodies were obtained from Cell Signaling Technology [pHSL Ser660 (Cat. 4126), pHSL Ser563 (Cat. 4139), HSL (Cat. #4107), pERK1/2 (Cat. 4370), and ERK 1/2 (Cat. 4695)], Santa Cruz Biotechnology [Actin (Cat. #SC69879)], Vala Biosciences [p-perilipin (Cat. 4854), perilipin (Cat. 4856)], and Sigma [M2 anti-FLAG antibody (Cat. F1804)]. The membrane was then rinsed with wash buffer and incubated for 1 h at room temperature in wash buffer containing 1% BSA with 1:2500 HRP-conjugated secondary antibody (Santa Cruz Biotechnology) or 1:2500 fluorophore-conjugated secondary antibody (Life Technologies). After further washing, membranes were visualized using enhanced chemiluminescence (Western Lightning, Perkin-Elmer) or imaged directly for fluorescent secondary antibodies and recorded on a BioRad ChemiDoc MP imaging station.
5
Western Blot Analysis of Cellular Signaling
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Cells or tissues were homogenized in lysis buffer (10 mM Tris–HCl, pH 7.6; 1% Triton X-100; 1 mM EDTA; 0.1% sodium deoxycholate) supplemented with protease and phosphatase inhibitors (Complete and PhosSTOP, Roche, Basel, Switzerland). Protein concentrations were determined by DC-Protein Assay (Bio-Rad, Hercules, CA, USA). Equal amounts were separated on SDS–polyacrylamide gels and transferred to 0.2 µm- PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked, incubated with primary antibodies and secondary antibodies (Merck-Millipore, Billerica, MA, USA or Dako, Glostrup, Denmark) afterwards. Primary antibodies used were against AKT, P-AKT (Ser473), GSK3β, P-GSK3β (Ser9), FAK, P-FAK (Tyr397), HSL, P-HSL (Ser660) (Cell Signaling Technology, Danvers, MA, USA), INTB1, P-INTB1 (Thr788/9) (Abcam, Cambridge, United Kingdom), Tubulin, Actin or GAPDH (Sigma-Aldrich, St. Louis, MO, USA). Immunoblots were detected by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific Waltham, MA, USA) and imaged with ImageQuant LAS 500 System (General Electric Healthcare, Little Chalfont, United Kingdom). Densitometries were measured using ImageJ software (NIH).
6
Western Blot Analysis of Signaling Proteins
7
Adipogenesis Regulation Protein Analysis
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5,5'dithiobis (2-nitrobenzoic acid), acetyl CoA, DLdithiothreitol (DTT), iodoacetamide, oxaloacetic acid, palmitoyl CoA, phenylmethylsulphonyl fluoride (PMSF) and triethanolamine hydrochloride were all purchased from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies anti-SIRT1, -PGC-1α, -NRF1, -FAS and -CD36 were obtained from Abcam (Cambridge, UK). Primary antibodies anti-FATP1 (SLC27A1), -NRG4 and -EPDR1 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies anti-p-AMPK (Thr 172), -total AMPK, -Acetylated lysine, -pHSL (Ser 660), -total HSL, -α-tubulin and -histone H3 were obtained from Cell Signalling Technology (Danvers, MA, USA). Primary antibodies anti-UCP1, -SIRT3, -GLUT4 and -ATGL were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
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