Plin1

Manufactured by Cell Signaling Technology

Sourced in United States

PLIN1 is a laboratory product manufactured by Cell Signaling Technology. It is a protein that plays a role in the regulation of lipid droplet formation and metabolism in cells.

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Anti-PLIN1 (3 citations) PLIN1) antibody (2 citations)

14 protocols using plin1

Comprehensive Western Blot Analysis

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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).

Kim M., Im S., Cho Y.K., Choi C., Son Y., Kwon D., Jung Y.S, & Lee Y.H. (2020). Anti-Obesity Effects of Soybean Embryo Extract and Enzymatically-Modified Isoquercitrin. Biomolecules, 10(10), 1394.

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Protein Expression Analysis of Adipose Tissue

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Cultured cells or the subcutaneous and epididymal adipose tissues of mice were lysed in radioimmunoprecipitation buffer (Beyotime Biotechnology, P0013B) containing protease and phosphatase inhibitors. Lysates were centrifuged at 13,000 g for 30 min at 4 °C. Protein concentrations of the extracts were determined with a Pierce BCA Protein Assay Kit (Thermo, 23227). Membranes were incubated with GAPDH (KangChen Biotech, KC-5G4), HSP90 (Cell Signaling Technology, 4877), ACTIN (Cell Signaling Technology, 4967), Cathepsin B (CTSB) (Cell Signaling Technology, 31718), PLIN1 (Cell Signaling Technology, CST, 9349s), SQSTM1 (Cell Signaling Technology, 5114), and autophagy antibodies (Cell Signaling Technology, 4445), including BECN1, LC3A/B, ATG5, ATG16L1, ATG7, and ATG3 overnight at 4 °C. The membranes were incubated with an appropriate secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. ECL Prime Western blotting Detection Reagent (GE Healthcare, RPN2232) was used to visualize protein bands by electro-chemoluminescence (ImageQuant LAS4000, USA).

Ju L., Han J., Zhang X., Deng Y., Yan H., Wang C., Li X., Chen S., Alimujiang M., Li X., Fang Q., Yang Y, & Jia W. (2019). Obesity-associated inflammation triggers an autophagy–lysosomal response in adipocytes and causes degradation of perilipin 1. Cell Death & Disease, 10(2), 121.

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Adipose Tissue Histological Analysis

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Epididymal adipose tissue sections were fixed in 4% PFA for three h and transferred to 70% ethanol at 4°C. Paraffin embedded sections were stained with H&E or immunolabeled with PLIN1 (Cell Signaling Technology, #3470, 1:1000 dilution), goat anti-rabbit IgG secondary (Abcam, Alexa Fluor® 647, #ab150079, 1:1000 dilution) and DAPI (Thermofischer, #62247, 1:4000 dilution). Slides were imaged using either a 10X objective on a Leica DMI8 widefield microscope and captured with a Leica DFC9000GT camera. Adipocyte sizing was performed as previously described (Cottam et al., 2022 (link)).

Winn N.C., Wolf E.M., Garcia J.N, & Hasty A.H. (2022). Exon 2-mediated deletion of Trem2 does not worsen metabolic function in diet-induced obese mice. The Journal of physiology, 600(20), 4485-4501.

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Murine Adipocyte Protein Extraction

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MEFs were harvested using the Qiagen kit with protease inhibitors. Samples were kept on ice for at least 20 minutes, after which non-soluble material was pelleted at 20,000 g for 20 minutes at 4°C. The supernatant was removed and protein concentrations were estimated using the BioRad Dc Protein assay.
For Western Blotting, 20 μg of the protein lysates were run on 4-12% Bis-Tris SDS-PAGE gels (NuPAGE Novex, Invitrogen) and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour at room temperature in 5 % milk in TBS-T, and incubated over night at 4°C with primary antibodies to FABP4 (Cell Signaling), PLIN1 (Cell Signaling), and HSC70 (Abcam, ab19136) as an internal control. Blots were washed in TBST and incubated with secondary antibody (HRP-anti-rabbit IgG for FABP4 and PLIN1, HRP-anti-rat IgG for HSC70, GE Healthcare) for 1 hour at room temperature. After washing, bands were visualised with enhanced chemiluminescence reagent (SuperSignal West Pico, Pierce) and X-Ray film. Protein density was analysed using ImageJ software (National Institutes of Health, USA).

Merkestein M., Laber S., McMurray F., Andrew D., Sachse G., Sanderson J., Li M., Usher S., Sellayah D., Ashcroft F.M, & Cox R.D. (2015). FTO influences adipogenesis by regulating mitotic clonal expansion. Nature Communications, 6, 6792.

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Visualization of Lipid Droplet Proteins

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CLSTN3B antibody was described previously (Zeng et al, PMID: 31043739); KDEL (Abcam, ab184819); PLIN1 (Abcam, ab3526); CGI-58 (Abcam, ab183739); UCP1(Abcam, ab209483); PDI (Cell signaling, C81H6); CS (Abcam, ab129095). Seipin (Cell signaling, 23846S), HA-tag (Cell signaling, 3724S), FLAG (Sigma, F1804), PLIN1 (Cell signaling, 9349S), p-HSL(s660) (Cell signaling, 4126), HSL (Cell signaling, 4107S), Alexa Fluor 546 Fluor Nanogold IgG goat anti-mouse (Nanoprobes, 7410–1ML), Alexa Fluor 488 Fluor goat anti-Rabbit (Invitrogen, A11034), Alexa Fluor 564 Fluor goat anti-mouse (Invitrogen, A11030), Alexa Fluor 405 Fluor goat anti-rabbit (Invitrogen, A31556).

Zhang C., Ye M., Melikov K., Yang D., Dias do Vale G., McDonald J., Eckert K., Lin M.J, & Zeng X. (2024). CLSTN3B enhances adipocyte lipid droplet structure and function via endoplasmic reticulum contact. bioRxiv.

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Analyzing Adipose Tissue Protein Profiles

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Water soluble proteins were isolated by pulverizing frozen adipose tissues in a lysis buffer purchased from Cell Signaling Technology Inc. (#9803, Danvers, MA, USA) containing both proteinase and phosphatase inhibitors (P8340, P5726 and P0044, Sigma). Protein concentrations were determined using a BCA assay kit (#P0010, Beyotime Biotechnology, Suzhou, China). Immunoblot analysis was performed as previously described [20 (link)]. Equal amounts of protein were resolved using SDS-PAGE gels. The following antibodies were used for detecting specific proteins: UCP1 (#ab23841, 1:1000), PGC-1α (#ab54481, 1:1000), OXPHOS (#ab110413, 1:1000), FIS1 (#ab71498, 1:1000) and OPA1 (#ab42364, 1:1000) were purchased from Abcam Inc. (Cambridge, MA, USA); COX IV (#4844, 1:1000), PLIN1 (#9349, 1:1000) and ATGL (2138, 1:1000) were from Cell Signaling Technology; NDUFS4 (O43181, 1:1000) was from Thermo Fisher Scientific; HSL (sc-25843, 1:1000), MGL (sc-398,942, 1:1000) and β-ACTIN (sc-47778, 1:3000) were obtained from Santa Cruz Biotechnology. Quantification of immunoreactive bands was carried out with the Image J software (National Institutes of Health, USA).

Shen W., Ren S., Hou Y., Zuo Z., Liu S., Liu Z., Fu J., Wang H., Yang B., Zhao R., Chen Y., Yamamoto M., Xu Y., Zhang Q, & Pi J. (2023). Single-nucleus RNA-sequencing reveals NRF1/NFE2L1 as a key factor determining the thermogenesis and cellular heterogeneity and dynamics of brown adipose tissues in mice. Redox Biology, 67, 102879.

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Immunoblotting Optimization for Cell Lysis

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For immunoblotting 2.5 × 10⁵ to 1 × 10⁶ cells were lysed in laemmli buffer containing 5% 2-β-mercaptoethanol (Sigma Aldrich, Germany), sonicated and boiled for 5 min at 95 °C. Proteins were size fractionated on prestained gradient polyacrylamide gels (Mini-PROTEAN^®TGX Stain-Free™ Precast Gels, Biorad, Germany), blotted onto 0.2 μm PVDF membrane, blocked 2 h in 5% low fat milk powder and incubated overnight with primary antibodies against ITGAV (BD Biosciences, Germany), ITGA5 (Biolegend, USA), AKT, phAKT (S473), phERK (Thr202/Tyr204), ERK, PLIN1, FABP4, YAP, TAZ (all from Cell signaling, USA), p21^Cip1 (ThermoScientific, Austria), survivin (Enzo Lifesciences, Switzerland), p73 (Imgenex, USA), p53 (MyBiosource, USA), GAPDH (6c5,Santa Cruz, Germany). After washing, horseradish peroxidase-conjugated sheep-anti-mouse and sheep-anti-rabbit antibodies (all from Cell signaling, USA) were incubated for 1 h and the reaction was visualized by enhanced chemiluminescence reagent ECL (Biorad, Germany) using a Biorad ChemidocMP gel analyzer for detection. Quantification was performed with the ImageLab 5.0 software (Biorad, Germany) according to the manufacturer’s instructions. Total protein loading was visualized using the ChemidocMP gel analyzer and employed as an internal loading control for all immunoblots59 (link).

Morandi E.M., Verstappen R., Zwierzina M.E., Geley S., Pierer G, & Ploner C. (2016). ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells. Scientific Reports, 6, 28889.

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Western Blot Analysis of Protein Markers

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Protein extracts were prepared as previously described^{22 (link)}. Overall, 10 μg of samples were electrophoresed and blotted to PVDF membrane (Bio-Rad, Hercules, CA, USA). Western blot was performed using primary antibodies against 15-lipoxygenase-1 (Mouse, Abcam, Cambridge, UK), PLIN1 (Rabbit, Cell Signaling, Danvers, MA, USA), Keratin 14 (Rabbit, Cell Signaling), phospho-RIP3(Rabbit, Abcam), RIP3(Rabbit, Cell signaling), ZBP1 (Rabbit, Cell Signaling), cleaved Caspase-3 (Asp175) (Rabbit, Cell Signaling), Caspase 3(Rabbit, Cell Signaling), Caspase 7(Rabbit, Cell Signaling), Tubulin (Cell Signaling), and β-actin (Mouse, Santa Cruz Biotechnology, Dallas, TX, USA) and secondary anti-mouse and anti-rabbit horse-radish peroxidase antibodies (Cell Signaling) as described previously. The blots were visualized with SuperSignal West Dura Substrate (ThermoFisher Scientific).

Kim S.N., Akindehin S., Kwon H.J., Son Y.H., Saha A., Jung Y.S., Seong J.K., Lim K.M., Sung J.H., Maddipati K.R, & Lee Y.H. (2018). Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice. Scientific Reports, 8, 8856.

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Adipocyte Lipolysis Regulation Assay

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Antibodies: pPKA and pPKC substrate specific antibodies; PLIN1 and pHSL (S660): from Cell Signaling Technology. CGI-58, HSL, GST and β-Actin: from Santa Cruz Biotechnology. Myc: from Roche. IP6K1: from Genetex. Plasmid construct: pT7T3D-PacI-PLIN1 construct was purchased from GE Healthcare/Open Biosystems. Reagents and Kits: Glycerol, FFA and TAG assay kits: from Cayman Chemicals; insulin Elisa kit from Crystal Chem; BCA protein assay kit: Pierce Biotechnology; Jetprime transfection reagent from Polypus; RetroX concentrator: from Clontech; Cyclic AMP from Cell Signaling; protein A/G beads from EMD Millipore. Pharmacologic modulators: Forskolin/FSK (Cell Signaling), isoproterenol (Sigma), CL316243 (Sigma); Rottlerin, LY333531 and GF109203X (Tocris). Protease plus phosphatase inhibitor tablets; Thermo Scientific, Waltham, MA. Unless otherwise stated, all the chemicals are purchased from Sigma Aldrich.

Ghoshal S., Tyagi R., Zhu Q, & Chakraborty A. (2016). Inositol hexakisphosphate kinase-1 interacts with perilipin1 to modulate lipolysis. The international journal of biochemistry & cell biology, 78, 149-155.

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Immunoblot Analysis of Adipogenic Regulators

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The primary antibodies used for this study were: PPARɣ (cat. no. CS2443, 1:50 dilution, Cell Signaling, Danvers, MA, USA), SREBP1c (cat. no. NB600-582, 1:8 dilution, Novus Biologicals, Littleton, CO, USA), FASN (cat. no. CS3189, 1:50 dilution, Cell Signaling), PLIN1 (cat. no. CS9349, 1:20 dilution, Cell Signaling), FABP4 (cat. no. AB92501, 1:50 dilution, Abcam, Cambridge, MA, USA), β-catenin (cat. no. NBPI-54467, 1:50 dilution, Novus Biologicals), β-actin (cat. no. MAB8919, 1:25 dilution, R&D Systems, Minneapolis, MN, USA), HSP60 (cat. no. F1800, 1:10 dilution, R&D Systems), and HSP70 (cat. no. 4872, 1:50 dilution, Cell Signaling). The secondary antibodies used for this study were: anti-rabbit HRP (cat. no. 040-656, 1:50 dilution & 042-206, no dilution, ProteinSimple), anti-mouse HRP (cat. no. 042-205, no dilution, ProteinSimple), anti-rabbit NIR (cat. no. 043-819, no dilution, ProteinSimple), and anti-mouse NIR (cat. no. 043-821, no dilution, ProteinSimple).

Urasaki Y, & Le T.T. (2022). Functional Complementation of Anti-Adipogenic Phytonutrients for Obesity Prevention and Management. Nutrients, 14(20), 4325.

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