HUVEC were lysed in 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl (Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), 0.25% sodium deoxycholate (Sigma-Aldrich), protease inhibitors (10 µg/mL Leupeptin, 10 µg/mL Aprotinin and 1 mM Phenylmethyl-Sulfonyl Fluoride, PMSF) (Sigma-Aldrich), and phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium vanadate, 5 mM sodium phosphate) (Sigma-Aldrich). Lysates (40 µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose sheets. Western Blot analysis was performed using antibodies against OPA1, DRP1, LC3 B-I/-II, BECLIN (Cell Signalling, Euroclone, Pero, Italy), CYPD, CPT1A, GLUT1 (Thermo Fisher Scientific), BNIP3 (Sigma-Aldrich), p62 (Invitrogen, Carlsbad, CA, USA), ATGL, PLIN2 and mitochondrial oxidative phosphorylation complexes (OXPHOS) (Abcam, Cambridge, UK). Actin (Santa Cruz, Dallas, Texas, USA) was the control of equal loading. After washing, secondary antibodies labelled with horseradish peroxidase (GE Healthcare, Waukesha, WI, USA) were used. Immunoreactive proteins were detected with Clarity™ Western ECL substrate by ChemiDoc MP Imaging System (Bio-Rad). Densitometry of the bands was performed with ImageJ. The Western blots shown are representative and the densitometric analysis was performed calculating the ratio between the protein of interest and Actin on three independent experiments ± Standard Deviation (SD).
Plin2
Manufactured by Abcam
Sourced in United States
PLIN2 is a protein that is a member of the perilipin family. It is involved in the regulation of lipid storage and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
Horseradish peroxidase, by GE Healthcare (1 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Oxphos, by Abcam (1 mentions)
Np 40, by Merck Group (1 mentions)
Beclin, by Cell Signaling Technology (1 mentions)
Cyp d, by Thermo Fisher Scientific (1 mentions)
Cpt1a, by Thermo Fisher Scientific (1 mentions)
Bnip3, by Merck Group (1 mentions)
Glut1, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Ds ri2, by Nikon (1 mentions)
Ds qi2, by Nikon (1 mentions)
Anti f4 80 antibody, by Abcam (1 mentions)
Eclipse ti2 microscope, by Nikon (1 mentions)
Gata3, by Santa Cruz Biotechnology (1 mentions)
Fv3000, by Olympus (1 mentions)
12 protocols using plin2
1
HUVEC Protein Expression Analysis
2
Adipose Tissue Macrophage Imaging
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6-wk-old C3(1)-TAg mice were fed an LFD or HFD for 6 wk. Mammary adipose tissue was isolated at 12 wk of age, a time point that coincides with DCIS (Holzer et al., 2003 (link)). Adipose tissue was sectioned (5 µm), stained with anti-F4/80 antibody (Abcam) to visualize ATMs, and counterstained with hematoxylin. Images were acquired with a Nikon Eclipse Ti2 microscope with the following setting: brightfield, objective magnification 20 and objective numerical aperture 0.45, room temperature, Color Camera Nikon DS-Ri2, and NIS-Element Version 5.02 software.
Mammary and visceral fat was obtained from WT and mNox2−/− mice fed a LFD or HFD for 10 wk. Adipose tissue was sectioned (5 µm) and stained with antibodies against MAC2 (Cedarlane) and PLIN2 (Abcam). Fluorescence images were acquired with a Nikon Eclipse Ti2 microscope with the following setting: objective magnification 20, objective numerical aperture 0.45, room temperature, emission wavelength of DAPI (457.5 nm), GFP (535.0 nm), and RFP (610 nm), Camera Nikon DS-Qi2, and NIS-Element Version 5.02 software.
Mammary and visceral fat was obtained from WT and mNox2−/− mice fed a LFD or HFD for 10 wk. Adipose tissue was sectioned (5 µm) and stained with antibodies against MAC2 (Cedarlane) and PLIN2 (Abcam). Fluorescence images were acquired with a Nikon Eclipse Ti2 microscope with the following setting: objective magnification 20, objective numerical aperture 0.45, room temperature, emission wavelength of DAPI (457.5 nm), GFP (535.0 nm), and RFP (610 nm), Camera Nikon DS-Qi2, and NIS-Element Version 5.02 software.
3
Immunofluorescence Staining of Microfluidic Cells
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Cells on the chip were fixed in 4% paraformaldehyde for 20 min at room temperature. Next, microchannels were washed with DPBS and filled with 0.2% Triton X‐100 for 10 min. Then, samples were blocked with goat serum for 1 h and incubated with primary antibody at 4°C overnight. Primary antibodies used in the experiment were listed as follow: CDH1 (mouse, CST, 1:100), CGB (rabbit, Abcam, 1:300), CK7 (mouse, Invitrogen, 1:100), GATA3 (mouse, Santa Cruz, 1:100), GLUT1 (rabbit, CST, 1:100), VE‐cadherin (rabbit, CST, 1:100), PLIN2 (rabbit, Abcam, 1:500). After washed with DPBS, samples were incubated with Alexa Fluor 488‐ or 594‐conjugated secondary antibodies (CST, 1: 500) at room temperature for 1 h and then stained with DAPI (CST, 1:4000) for 10 min. For F‐actin staining, samples were stained with Alexa Fluor 488‐conjugated phalloidin (Biotium) for 20 min at room temperature according to the manufacture' s instruction. The stained samples were imaged using a confocal microscope (FV3000, Olympus).
4
Quantification of Crown-like Structures and Caspase-3 Activity in Epididymal Fat
5
Stimulating Lipid Droplet Accumulation in Rat Hepatocytes
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Rat hepatocytes were isolated by collagenase perfusion of the liver as described elsewhere99 . Cells were cultured for 24 h in DMEM with 10% FBS. Prior stimulation, cells were incubated in serum free DMEM overnight.
To assess lipid droplet accumulation, primary rat hepatocytes were stimulated with complete DMEM medium supplemented with HSP70 (15 ng/ml) or GRP78 (3 ng/ml) or Oleic acid (400 µmol/L) for 24 h. To test the effect of TLR4 inhibition, cells were pretreated with or without TAK-242 (200 nM) for one hour. After the stimulation, half of the cells were stained with Nile red (100 ng/mL) for 45 min. Stained cells were used for immunofluorescence to quantify the deposition of lipid droplets. Unstained cells were used to assess lipid uptake and liponeogenesis through quantitative real-time PCR. Primer sequences are reported in Supplementary TableS6 .
Images of lipid droplets were taken using confocal microscope Nikon A1 RHD25 and NIS-Elements imaging software was used to analyze images.
DMEM and FBS were obtained from Corning (New York, NY), Oleic acid and Nile red were obtained from Merck (Darmstadt, DE) and Plin2 was obtained from Abcam (Cambridge, UK).
To assess lipid droplet accumulation, primary rat hepatocytes were stimulated with complete DMEM medium supplemented with HSP70 (15 ng/ml) or GRP78 (3 ng/ml) or Oleic acid (400 µmol/L) for 24 h. To test the effect of TLR4 inhibition, cells were pretreated with or without TAK-242 (200 nM) for one hour. After the stimulation, half of the cells were stained with Nile red (100 ng/mL) for 45 min. Stained cells were used for immunofluorescence to quantify the deposition of lipid droplets. Unstained cells were used to assess lipid uptake and liponeogenesis through quantitative real-time PCR. Primer sequences are reported in Supplementary Table
Images of lipid droplets were taken using confocal microscope Nikon A1 RHD25 and NIS-Elements imaging software was used to analyze images.
DMEM and FBS were obtained from Corning (New York, NY), Oleic acid and Nile red were obtained from Merck (Darmstadt, DE) and Plin2 was obtained from Abcam (Cambridge, UK).
6
Western Blot Analysis of HUVECs
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HUVECs were lysed in western and IP buffer (Beyotime, P0013) containing proteinase inhibitor PMSF (Sigma-Aldrich). After centrifuging at 12,000 × g, 4°C for 15 min, the supernatant was collected. Protein samples were loaded on 12% or 9% SDS/PAGE at 4°C and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, IPVH00010). The membranes were blocked with 5% nonfat milk for 1 h at room temperature, incubated with primary antibodies: LAMP1(Cell Signaling Technology, 9091); P21(Cell Signaling Technology, 2947); PLIN2 (Abcam, ab-108323); ACTB (Sigma-Aldrich, 122M4782); GAPDH (Santa Cruz Biotechnology sc-47724) at 4°C overnight, then horseradish peroxidase-linked secondary antibodies (Jackson ImmunoResearch, goat anti-rabbit:13963, goat anti-mouse:130389). Fluorescence signals were detected with X-ray films after being incubated with Immobilon Western Chemiluminescent HRP substrate for 3 min. The relative quantity of proteins was quantified by ImageJ software.
7
Western Blot Analysis of Metabolic Proteins
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Cells were lysed in protein lysis buffer (1% NP-40, 0.5% Triton, 10% glycerol, 150 mM NaCl, 1 mM EDTA, 0.5 M Tris) with complete protease and phosphatase inhibition cocktail tablets (Roche, Penzberg, Germany) and protein content was measured by BCA asay (ThermoFisher Scientific). 20 µg of protein were separated by 4–12% Nupage Bis-Tris gel (ThermoFisher) and wet transferred on ice to nitrocellulose membranes for 1 hr at 100 Volts. In some cases, the gels were cut and strips from multiple gels were transferred together41 (link). Membranes were blocked with 5% milk in TBST and incubated overnight at 4 °C with antibodies recognizing PLIN2 (1:1000, Abcam, Cambridge, MA)7 (link), GAPDH (1:1000, Millipore)42 (link), phosphorylated or total AMPK43 (link),44 and ACC45 (link),46 (link) (Cell Signaling, Beverly, MA). Signal was detected with enhanced chemiluminescence (GE Healthcare, Piscataway, NJ) following incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz Technology). Quantitation of Western films was performed using ImageJ software and results were normalized to GAPDH.
8
Immunoblotting with Chemiluminescent Substrates
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Immunoblotting with Immunostar LD chemiluminescent substrates (290–69904, Wako) was performed. Signals were detected with an LAS-3000 image analyser (Fujifilm, Tokyo, Japan) as previously described49 (link),50 (link). Lamin β1 (LMNB1; PM064, 1:10000) was purchased from MBL (Nagoya, Japan). CTSB (ab58802, 1:400), CIDEC/FSP27 (ab16760, 1:2000), PLIN1 (ab61682, 1:400) and PLIN2 (ab108323, 1:1000) were purchased from Abcam (Cambridge, UK). FLAG M2 (F1804, 1:5000) was purchased from Sigma-Aldrich. PPARG (sc-7273, 1:2000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (010-25521, 1:5000) was purchased from Wako.
9
Western Blot Analysis of Lipid Metabolism Proteins
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CGI-58 and Plin-2 antibodies were obtained from Abcam. β-actin, Phospho-JNK (Thr183, Tyr185), JNK, Phospho-S505-cytosolic phospholipase A2 (cPLA2), and cPLA2 antibodies were purchased from Cell Signaling Technology Co. Lysis buffer (2% SDS, 62.5 mM Tris–HCl pH 6.8, and 10% glycerol) was used to lyse the cells. Subsequently, 20 μg protein of each sample was electrophoresed and separated by SDS-PAGE. Then, the proteins were transferred to PVDF membranes (Millipore Corporation, United States). The membrane was incubated in 5% of defatted milk for 1 h at room temperature. The membrane was incubated with the primary antibodies overnight at 4 °C. After incubation, the membrane was washed in PBST three times and subsequently incubated with HRP-conjugated secondary antibodies. Immobilon Western Chemiluminescent HRP substrate (Millipore Corporation, United States) was used to obtain visualization of target protein bands.
10
Western Blot Analysis of TBE Proteins
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TBE sample was separated by electrophoresis on 4–12% sodium dodecyl sulfate–polyacrylamide gels and then transferred electrophoretically to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then washed three times with 0.1% Triton X-100 (TBSX). The membranes were then gently agitated and incubated at 4 °C overnight with the following primary antibodies: HT7 (Thermo Fisher, MN1000, 1:500), AT8 (Thermo Fisher, MN1020, 1:1000), and Plin2 (Abcam, ab52356, 1:1000). The following day, the membranes were washed and then incubated with horseradish peroxidase-labeled anti-mouse or anti-rabbit secondary antibodies for 1 h at RT. Subsequently, membrane-bound horseradish peroxidase-labeled antibodies were detected using an enhanced chemiluminescence detection system including the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Quantification of the bands was performed by ImageJ 1.44 (Image Processing and Analysis in Java).
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