Eight days after induction of differentiation the 3T3-L1 cells were fixed with 4% formaldehyde and stained overnight with oil red O (Sigma-Aldrich) as previously described.35 (link),36 (link) For Western analysis, cells were washed in PBS and lysed in modified RIPA buffer, 50 mm Tris (pH 7.4), 150 mM NaCl, 1% IGEPAL, 5 mM EDTA, and protease inhibitor cocktail (Sigma-Aldrich). The lysate was cleared by centrifugation. Equal amounts of protein (10–30 µg) were resolved by SDS-PAGE and transferred to polyvinylidene membrane (Bio-Rad) for western analysis with the primary antibodies for aP2 (adipocyte fatty acid binding protein 4 [FABP4]) (R&D), adipsin (P-16) (Santa Cruz), and β-actin (13E5) (Cell Signaling). Signal intensities of western blots were imaged using the BioRad ChemiDoc Imager and quantified using Bio-Rad Image Lab software. For graphical analysis, data are presented as an average protein induction from at least three independent experiments.
Polyvinylidene membrane
Manufactured by Bio-Rad
Sourced in United States
Polyvinylidene membrane is a type of microporous membrane used in various laboratory applications. It is made from polyvinylidene fluoride (PVDF) material and is characterized by its chemical and thermal resistance, as well as its high mechanical strength. The core function of the polyvinylidene membrane is to provide a durable and reliable medium for various separation and filtration processes in research and analytical environments.
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Lab products found in correlation
Image lab software, by Bio-Rad (3 mentions)
Irdye 700, by LI COR (2 mentions)
Irdye 800, by LI COR (2 mentions)
Beyoecl plus, by Beyotime (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
β actin 13e5, by Cell Signaling Technology (1 mentions)
Oil red o, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Odyssey infrared imaging system and software, by LI COR (1 mentions)
Pathscan rtk signaling antibody array kit, by Cell Signaling Technology (1 mentions)
Anti bax, by Bioss Antibodies (1 mentions)
Anti p53, by Wanlei (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti bcl 2, by Wanlei (1 mentions)
Myd88, by Abcam (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
14 protocols using polyvinylidene membrane
1
Adipocyte Differentiation Assay
2
Western Blot Analysis of RTK Signaling
3
Apoptosis Signaling in Granulosa Cells
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Granulosa cells were incubated with 3-NPA at 5.0 mmol/l for 24 h and then were harvested. The total protein was extracted using RIPA Lysis Buffer and 10% phenylmethanesulfonyl fluoride (Beyotime, China). Protein concentration was determined by using a BCA Protein Assay Kit (Beyotime, China). Approximately, the same amount of protein was separated using 10–12% SDS-PAGE, transferred electrophoretically onto the polyvinylidene membrane (Bio-Rad) and blocked with 5% nonfat dry milk. The primary antibodies used in the present study were anti-Caspase 3 (CST, U.S.A.), anti-Bcl-2 (Wanlei Biotechnology, China), anti-p53 (Wanlei Biotechnology, China), anti-Bax (Bioss Biotechnology, China), anti-β-actin (TransGen Biotechnology, China). The membrane was incubated with the primary antibody solution overnight at 4°C, and then washed four times with the TBST (TBS, 0.1% Tween 20). The corresponding secondary antibody (1:5000) was added and incubated at room temperature for 1 h. The protein bands were visualized by using the BeyoECL Plus (a chemiluminescence reaction; Beyotime, China) in an Image Lab software (Bio-Rad, U.S.A.). The bands were quantified using an ImageJ software (NIH, U.S.A.).
4
Western Blot Analysis of Apoptosis Markers
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The rat heart tissue and cultured H9c2 cell line were homogenized in RIPA buffer. Proteins from the homogenate (20 μg/well) were separated on a one-dimensional 4%–10% SDS polyacrylamide gel and transferred to a polyvinylidene membrane (Bio-Rad, Hercules, CA, USA) with a semidry electrotransfer apparatus for 1.5 h at 300 mA. The expression levels of Myd88, c-Myc, cleaved-Bid, p53, Bax, Bcl-2, cleaved-caspase-3, Irak-2, and IL-1R were determined (Myd88, c-Myc, p53, Bax, Bcl-2, and IL-1R; 1:1,000; Abcam, Cambridge, MA, USA; Irak-2; 1:1,000; Cell Signaling Technology, Danvers, MA, USA; cleaved Bid and cleaved-caspase-3; 1:500; WanleiBio, Shenyang, Liaoning Province, China). The immunoblots were visualized by enhanced chemiluminescence (ECL detection kit from GE Healthcare, Chicago, IL, USA).
5
Western Blot Analysis of Post-SCI Immune Response in Rats
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At the post-SCI 7day, rats were randomized selected to assess their immunity. Another group of examiners blinded to the treatment groups processed the western blotting. Tissues were washed three times with ice-cold PBS and lysed in RIPA buffer containing 1% PMSF. After leaving the samples on ice for 30 min, they were centrifuged at 12,000g for 15 min at 4 °C. Protein concentrations in the supernatant were assessed using a Micro BCA Protein Assay kit. Equal amounts of protein extracts were resolved by 10–12% SDS-PAGE and electrotransferred onto a polyvinylidene membrane (Bio-Rad, USA). After blocking in Tris-buffered saline plus 5% (w/v) milk, the membranes were exposed to primary antibodies overnight at 4 °C. The samples were incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. An independent examiner blinded to the treatment groups assessed these gene expressions. Signals were visualized by a ChemiDoc Touch Imaging System (Bio-Rad, USA), and ImageJ software was used to assess their average inflorescence intensity. All experiments were repeated three times.
6
NF-κB Protein Quantification in PBMCs
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Determination of NF-κB protein was performed with the protein extract from PBMCs (n = 1 volunteer) by Western blotting. The PBMCs were incubated with or without LPS (1 µg/mL) and BRE (0 or 50 µg/mL) for 1 h. Thirty micrograms of the proteins were separated by electrophoresis in a 10% polyacrylamide gel and transferred at 4 °C to a polyvinylidene membrane (Biorad). Immunoblots were blocked with TBS–Tween-20 0.1% containing 5% dry milk, and then probed with a primary antibody (NF-κB p65, Cell Signaling Technology, Leiden, The Netherlands). The immunoblots were incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). The immune reactive strips were visualized by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific). Band densities were quantified using Fiji [15 (link)]. β-actin (Cell Signaling Technology) was used as an internal control on each gel to normalize signal intensities between gels.
7
Immunoblot Protein Detection Protocol
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For immunoblots, proteins were extracted as described in [49 (link)], and 50 μg of cell extract were resolved on a 10% polyacrylamide-SDS gel before being transferred onto a polyvinylidene membrane (BioRad, Hercules, CA). All primary antibodies were purchased from Cell Signaling Technologies (Beverly, MA), except for the PIM1 antibody, which was purchased from Abcam (Cambridge, MA). All membranes were probed with primary antibodies overnight at 4°C, then incubated with Alexa Fluor-labeled secondary antibodies (IRDye 680LT goat-anti mouse, IRDye 800CW goat-anti rabbit (LI-COR, Lincoln, NE)) for 1h and scanned with the Odyssey system (LI-COR, Lincoln, NE). The secondary antibodies, IRDye700 and IRDye800, and the Odyssey Infrared Imaging System used to image the membranes were obtained from Li-Cor (Lincoln, NE).
8
Western Blot Analysis of BRF2 Protein
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Equal amounts of protein were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and transferred to polyvinylidene membrane (Bio-Rad, Shanghai, China).
After blocking in 3% bovine serum albumin in phosphate-buffered saline with 0.05%
Tween-20, the membranes were incubated with rabbit polyclonal anti-BRF2 antibody (1:1000;
ab154658; Abcam, Shanghai, China) and rabbit monoclonal anti-glyceraldehyde-3-phosphate
dehydrogenase antibody (1:5000; ab181602; Abcam). The secondary antibodies are horseradish
peroxidase conjugated. The blots were visualized with chemiluminescence.
electrophoresis and transferred to polyvinylidene membrane (Bio-Rad, Shanghai, China).
After blocking in 3% bovine serum albumin in phosphate-buffered saline with 0.05%
Tween-20, the membranes were incubated with rabbit polyclonal anti-BRF2 antibody (1:1000;
ab154658; Abcam, Shanghai, China) and rabbit monoclonal anti-glyceraldehyde-3-phosphate
dehydrogenase antibody (1:5000; ab181602; Abcam). The secondary antibodies are horseradish
peroxidase conjugated. The blots were visualized with chemiluminescence.
9
Quantitative Western Blot Analysis of Hypothalamus
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Hypothalamus tissue was lysed in RIPA buffer and protein concentration was determined by BCA assay. Equal amount of proteins (approximately 30 µg) per sample were separated using 10% SDS-PAGE, transferred electrophoretically onto the polyvinylidene membrane (Bio-Rad), and blocked with 5% nonfat dry milk in Vertical Electrophoresis Systems (Bio-Rad). The primary antibodies used in this study were OAZ1 (A744, 1:1,000 dilution; ABclonal, Wuhan, China), SSAT (10708-1-AP, 1:500 dilution; Proteintech, Wuhan, China), and β-actin (A3854, 1:50,000 dilution; Sigma-Aldrich). The membrane was incubated with the primary antibody solution overnight at 4°C and then washed with the TBST. The corresponding secondary antibody (A028, 1:1,000 dilution; Beyotime, Shanghai, China) was added and incubated at room temperature for 2 h. The protein bands were visualized by using the BeyoECL Plus (a chemiluminescence reaction; Beyotime, China) in Gel Imaging Systems with an Image Lab software (Bio-Rad, USA). The bands were quantified using an ImageJ software (NIH, USA).
10
Foxf2 Protein Expression Analysis
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After transfection, 10 6 cells were lysed in RIPA buffer (Thermo Fisher Scientific, USA) and were centrifuged at 12,000 g for 5 min. Supernatants were extracted to purify total proteins, which were quantified using the bicinchoninic acid assay. Protein samples were diluted to equal amounts and were separated by SDS-PAGE at 120 V for 2 h. The separated proteins were then transferred to a polyvinylidene membrane (Bio-rad, USA) at 15 V for 30 min and were blocked with 10% defatted milk powder at 37°C for 2 h. Rabbit anti-human Foxf2 polyclonal antibody (1:1000) or GAPDH (1:1000) at 20 µL final volume was incubated at 4°C overnight. After this, the membrane was rinsed three times (10 min each) with Tris-buffered saline-Tween-20 at 25°C. Goat anti-rabbit secondary antibody, at 20 µL final volume, was then added for 1-hour incubation, followed by the enhanced chemiluminiscence chromogenic substrate. Quantity One software (Bio-rad, USA) was employed to analyze the blotting bands, in which the ratio of intensity of target protein to that of GAPDH bands was calculated to reflect the relative protein level.
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