The actin segmentation analysis was done using a method described previously [14 (link)]. Briefly, cells were lysed directly using actin stabilization buffer (50 mM PIPES (pH 6.9), 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 2 mM ATP, 5% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% Tween 20, 0.1% β-mercaptoethanol, 1:100 protease inhibitor mixture, and 1:100 phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). The cells were collected into tubes, homogenized with a 28G syringe, and incubated at 37 °C for 10 min, followed by centrifugation at 300 × g at room temperature to remove insoluble particles. The lysate was centrifuged at 100,000 × g at 37 °C for 1 h to precipitate F-actin and to separate the G-actin that remained soluble in the supernatant. The pellet containing F-actin was resuspended and dissociated with 10 µM cytochalasin D (Sigma-Aldrich, St. Louis, MO, USA). Both fractions were resolved by SDS-PAGE and probed with an anti-β-actin antibody. The ratio of F-actin over G-actin was determined and analyzed by Student’s t-test.
Phosphatase inhibitor mixture
Manufactured by Merck Group
Sourced in United States
The Phosphatase inhibitor mixture is a laboratory reagent designed to inhibit the activity of phosphatases, a class of enzymes that remove phosphate groups from proteins and other molecules. This product is commonly used in biochemical research to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor mixture, by Merck Group (10 mentions)
Protease inhibitor mixture, by Roche (8 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Ab23841, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
β actin mouse mab, by Merck Group (2 mentions)
Alexa fluor 680 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab152, by Merck Group (2 mentions)
Image studio software, by LI COR (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Ab10983, by Abcam (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
V7931, by Promega (1 mentions)
Protease inhibitor, by Roche (1 mentions)
28 protocols using phosphatase inhibitor mixture
1
Quantifying Cellular Actin Dynamics
2
Cell Lysis and Protein Analysis
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Cells were lysed in M2 lysis buffer (150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 5 mM EDTA, 1% Nonidet P-40) containing a protease inhibitor mixture (Roche Applied Science) and a phosphatase inhibitor mixture (Sigma, MO, USA). Equal amounts of total protein were subjected to SDS-PAGE analysis and immunoblotting with the appropriate antibodies.
3
Immunohistochemical and Immunoblotting Analysis of Beige Adipocyte Markers
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Adipose tissues were fixed in 10% neutral formalin, embedded in paraffin, cut into 5 μm sections, and stained with UCP1 antibody (1:150, abcam, ab10983) to detect UCP1 protein expression and beige adipocyte formation as we described previously described (Zha et al. 2015 ; Nguyen et al. 2017 ). Tyrosine hydroxylase (TH) and UCP1 protein expression in IWAT and IBAT was measured by immunoblotting as we described (Zha et al. 2015 ; Nguyen et al. 2017 ). Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer containing 50 mmol/L Tris‐HCl, 1 mmol/L EDTA, 1% Nonidet P‐40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 200 mmol/L Na3VO3, 1% protease inhibitor mixture (Sigma), and 1% phosphatase inhibitor mixture (Sigma). Tissue lysates were separated using SDS‐PAGE. Proteins on the gels were transferred to nitrocellulose membrane (Bio‐Rad, Hercules, CA). The transferred membranes were blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680‐conjugated secondary antibodies (Life Science Techenologies). The blots were developed with a Li‐COR Imager System (Li‐COR Biosciences, Lincoln, NE). The following primary antibodies were used: UCP1 (1:500, abcam, ab23841), TH (1:1000, Millipore, AB152), and α‐Tubulin (1:1000, Advanced BioChemicals, ABCENT4777).
4
Protein Extraction and Western Blot Analysis
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The cells were harvested with M-PER mammalian protein extraction reagent (Cat#78501, Thermo Scientific, Rockford, IL) supplemented with protease inhibitor mixture (Cat#P8340, Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor mixture (Cat#P-0044, Sigma-Aldrich, St. Louis, MO). Cell lysates were analyzed using Western blot procedures described in details in the Supplementary Methods.
5
In Vitro Kinase Assay for Gad8
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We applied a nonradioactive in vitro kinase assay for Gad8 [59 (link)], based on the use of GST-Fkh2 as a substrate [37 (link)]. For the Gad8 kinase assay, a DNA fragment encoding amino acid residues 291 (Gln) to 411 (Pro) of Fkh2 was expressed in Escherichia coli BL21 strain as GST fusion, using the pGEX-4T1 expression vector and purified. Cells expressing Gad8-HA extracts were immunoprecipitated, and the resultant immunocomplexes were resuspended in 30 μl of kinase buffer (10 mM MgAc, 100 mM ATP, and phosphatase inhibitor mixture, Sigma) containing 0.1 μg of GST-Fkh2. After incubation for 10 min at 30°C, the reaction was terminated by addition of 7 μl of 5 × SDS-PAGE sample buffers and incubated for 5 min at 80°C. The reaction was detected by Western blot analysis using anti-phospho-AKT substrate antibody (Cell Signaling Technology).
6
Mosquito Protein Extraction and Western Blot
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Mosquito tissues collected from individual mosquitoes were separately put into micro-centrifuge tubes containing 100μL of breaking buffer [50 mM Tris (pH 7.4), 1% IGEPAL, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethyl-sulfonylfluoride, 1X protease inhibitor mixture, and 1X phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, Missouri, USA)] and homogenized using a pellet pestle. The homogenates were centrifuged at 13,000 rpm for 5 min. The supernatants were transferred into a Qiashredder Column (Qiagen, Los Angeles, California, USA) and centrifuged again under the same conditions for 10 min. The flow-through was transferred to a clean micro-centrifuge tube to conduct a Western blot analysis using anti-phosphoric JNK antibody (V7931, Promega) and anti-JNK antibody (sc-571, Santa Cruz). The blot was developed by VisGlow Chemiluminescent Substrate and HRP (Visual Protein).
7
Western Blot Analysis of Phospho-STAT5 and JAK3
8
Immunoblotting for Protein Detection in Fat Tissues
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Immunoblotting for protein detection was conducted as we described [27 (link),32 (link)]. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Tissue lysates were resolved by SDS-PAGE gels. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies used were as follows: Anti-UCP1 antibody (1:500, ab23841, Abcam, Cambridge, MA, USA); Anti-α-Tubulin antibody (1:1000, ABCENT4777, Advanced BioChemicals, Lawrenceville, GA, USA); Mitochondrial total OXPHOS protein antibody set (Abcam,ab110413); and Anti-pHSL (4126s, Cell Signaling Technology, Danvers, MA, USA); DNMT3b (sc-393845, Santa Cruz, Dallas, TX, USA, sc-393845); HSL (Cell Signaling, 4107s).
9
Western Blot Analysis of Protein Signaling
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Cells were lysed with cell lysis buffer (Cell Signaling Technology) containing protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor mixture (Sigma-Aldrich) for 30 min on ice. The samples were centrifuged at 14,000 rpm for 15 min at 4°C to remove cell debris. Protein concentration was determined using a BCA protein assay kit (Pierce). Proteins were subjected to SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk in TBS solution containing 0.05% Tween-20 for 1 h at room temperature. Then the membrane was incubated with antibodies against GAPDH (1:1,000), IκB (1:500, Cell signaling pathway), and pIκB (1:500, Cell signaling pathway) overnight at 4°C. After washing three times with TBST solution, the membrane was incubated with HRP-conjugated goat anti-rabbit (1:2,000; Zymed) for 1 h at room temperature. Immuno-reactive bands were detected using enhanced chemiluminescence (Thermo Scientific) and captured with an Odyssey Fc Imager (Li-cor biosciences Inc.). Western blot data were analyzed with ImageJ software.
10
Immunoblotting of Adipose Tissue Proteins
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Protein expression in adipose tissue was measured by immunoblotting as we described69 (link),76 (link),77 (link). Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Tissue lysates were resolved by SDS-PAGE. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (Life Science Technologies). The blots were developed with a Li-COR Imager System and analyzed with Li-COR Image Studio Software (version 2.1, Li-COR Biosciences, Lincoln, NE). The following primary antibodies were used: UCP1 (1:1000, Abcam, ab23841), TH (1:1000; AB152, EMD Millipore, Temecula, CA), NT-3 (1:1000, AF-267-NA, R&D Systems), pHSL (1:1000, 4126S, Cell Signaling Technology), HSL (1:1000, 4107S, Cell Signaling Technology), and α-tubulin (1:1000, Advanced BioChemicals, ABCENT4777). The antibody information is listed in Supplementary Table 1 .
Tissue and serum NT-3 content was measured by ELISA (ABCE-EL-M2438, Advanced BioChemicals, Lawrenceville, GA).
Tissue and serum NT-3 content was measured by ELISA (ABCE-EL-M2438, Advanced BioChemicals, Lawrenceville, GA).
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